Systems and methods for multicolor imaging

ABSTRACT

Disclosed herein, inter alia, are methods and systems of image analysis useful for rapidly identifying and/or quantifying features.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/703,720, now U.S. Pat. No. 11,415,515, filed Aug. 6, 2021, which is acontinuation of PCT Application No. PCT/US21/64423, which claims thebenefit of U.S. Provisional Application No. 63/128,477, filed Dec. 21,2020, each of which are incorporated herein by reference in theirentirety and for all purposes.

BACKGROUND

Next generation sequencing (NGS) methods typically rely on the detectionof genomic fragments immobilized on an array. For example, insequencing-by-synthesis (SBS), fluorescently labeled nucleotides areadded to an array of polynucleotide primers and are detected uponincorporation. The extension of the nucleic acid primer along a nucleicacid template is monitored to determine the sequence of nucleotides inthe template. Each detection event, (e.g., a feature), can bedistinguished due to their location in the array.

For these and other applications of polynucleotide arrays, improvementshave recently been made to increase the density of features in thearrays. For example, such arrays have at least 50,000 features/cm²,100,000 features/cm², 1,000,000 features/cm², 5,000,000 features/cm², orhigher. Technological advances reduced the typical distance betweenneighboring features such that the features are only slightly largerthan the optical resolution scale, the pixel pitch of the camera, orboth. High resolution optics (e.g., objectives with a 0.8 numericalaperture (NA)), combined with fast imaging methods described hereinenable increased analysis and throughput in imaging systems.

BRIEF SUMMARY

Disclosed herein, inter alia, are solutions to the aforementioned andother problems in the art. This disclosure provides methods and systemsof image analysis useful for rapidly identifying and/or quantifyingfeatures on a substrate.

In an aspect, an imaging system is provided. In non-limiting exampleembodiments, the imaging system includes a plurality of sensor arrays(e.g., two or four independent time delay integration [TDI] sensorarrays), two light sources (e.g., two lasers) that illuminate a sample,a first optical system that directs one excitation beam from each lightsource onto a sample, a second optical system that directs fluorescentemissions from the sample to each sensor array.

In another aspect, there is disclosed an imaging system including: asample stage moving at a sample stage speed, wherein the sample stage isconfigured to receive a sample including a first fluorophore and asecond fluorophore; a first sensor array and a second sensor array; afirst light source configured to provide a first excitation beam and asecond light source configured to provide a second excitation beam; afirst optical system configured to direct a first excitation beam andsecond excitation beam onto a sample, wherein the interaction of thefirst excitation beam with the first fluorophore generates a firstfluorescent emission, and the interaction of the second excitation beamwith the second fluorophore generates a second fluorescent emission; asecond optical system configured to direct the first fluorescentemission to the first sensor array, and the second fluorescent emissionto the second sensor array, wherein the first fluorescent emissionimpinges upon and generates a first charge that travels across the firstsensor array, wherein the second fluorescent emission impinges upon andgenerates a second charge that travels across the second sensor array;and wherein the travel of at least one of the first and the secondcharge is synchronized with the sample stage speed. In embodiments, theimaging system further includes a third fluorophore, and a fourthfluorophore; a third sensor array, and a fourth sensor array, whereinthe interaction of the first excitation beam with the third fluorophoregenerates a third fluorescent emission, and the interaction of thesecond excitation beam with a fourth fluorophore generates a fourthfluorescent emission; wherein the second optical system is configured todirect the third fluorescent emission to the third sensor array and thefourth fluorescent emission to the fourth sensor array, wherein thethird fluorescent emission impinges upon and generates a third chargethat travels across the third sensor array, wherein the fourthfluorescent emission impinges upon and generates a fourth charge thattravels across the fourth sensor array, wherein the travel of at leastone of the third and the fourth charge is synchronized with the samplestage speed.

In another aspect, there is disclosed an imaging system including: asample stage moving at a sample stage speed, wherein the sample stageincludes a sample including a first fluorophore, a second fluorophore, athird fluorophore, and a fourth fluorophore; a first sensor array,second sensor array, third sensor array, and a fourth sensor array; afirst light source configured to provide a first excitation beam and asecond light source configured to provide a second excitation beam; afirst optical system configured to direct a first excitation beam andsecond excitation beam onto a sample, wherein the interaction of thefirst excitation beam with the first fluorophore generates a firstfluorescent emission, the interaction of the second excitation beam withthe second fluorophore generates a second fluorescent emission, theinteraction of the first excitation beam with a third fluorophoregenerates a third fluorescent emission, and the interaction of thesecond excitation beam with a fourth fluorophore generates a fourthfluorescent emission; a second optical system configured to direct thefirst fluorescent emission to the first sensor array, the secondfluorescent emission to the second sensor array, the third fluorescentemission to the third sensor array, the fourth fluorescent emission tothe fourth sensor array, wherein the first fluorescent emission impingesupon and generates a first charge that travels across the first sensorarray, wherein the second fluorescent emission impinges upon andgenerates a second charge that travels across the second sensor array,wherein the third fluorescent emission impinges upon and generates athird charge that travels across the third sensor array, wherein thefourth fluorescent emission impinges upon and generates a fourth chargethat travels across the fourth sensor array, wherein the travel of atleast one of the first, the second, the third and the fourth charge issynchronized with the sample stage speed.

In another aspect, there is disclosed a method of imaging a sampleincluding: a) directing a first excitation beam and a second excitationbeam onto a sample, wherein the sample is on a sample stage moving at asample stage speed, wherein the sample comprises a first fluorophorethat generates a first fluorescent emission and a second fluorophorethat generates a second fluorescent emission following interaction witha first excitation beam and a second excitation beam, respectively; b)directing the first fluorescent emission to impinge upon and generates acharge that travels across a first sensor array at a first charge speed,and directing the second fluorescent emission to impinge upon andgenerate a second charge that travels across a second sensor array at asecond charge speed, wherein at least one of the first charge speed andthe second charge speed is synchronized with the sample stage speed; andc) scanning the sample in a scan dimension and repeating step a) andstep b) to form an image of the sample. In embodiments, the samplefurther includes a third fluorophore that generates a third fluorescentemission and a fourth fluorophore that generates a fourth fluorescentemission following interaction with a first excitation beam and a secondexcitation beam, respectively; and directing said third fluorescentemission to impinge upon and generate a third charge that travels acrossa third sensor array at a third charge speed, and directing said fourthfluorescent emission to impinge upon and generate a fourth charge thattravels across a fourth sensor array at a fourth charge speed.

The details of one or more variations of the subject matter describedherein are set forth in the accompanying drawings and the descriptionbelow. Other features and advantages of the subject matter describedherein will be apparent from the description and drawings, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary illustration of a 4-color imaging system usingone camera, wherein the camera contains four sensor arrays (i.e., imagesensor arrays); and

FIG. 2 is an exemplary illustration of a 4-color imaging system usingtwo cameras, wherein each camera contains two sensor arrays (i.e., imagesensor arrays).

DETAILED DESCRIPTION

Nucleic acid sequencing, biomolecule imaging, and other uses ofbiochemical arrays require advanced imaging systems to achievecommercially viable data acquisition rates. The number of biochemicalexperiments from which data may be collected per unit time can depend onthe array density and image acquisition speed, among other factors.Increased array density can complicate the image acquisition problembecause it can make keeping track of the identity of each experiment inan image challenging. One of most common methods of sequencing DNA is tofluorescently label each of the four nucleotide bases of a DNA strand.Typically, the resolution of each base pair is about 0.5 nm across, andthe light from their fluorescence is extremely weak. High-sensitivityimage sensors and imaging systems are needed.

Image acquisition speed can be increased by using multiple excitationbeams to image various fluorophores in the sample (e.g., sample thatincludes nucleic acid for sequencing). However, imaging using multipleexcitation beams may require additional optical elements (e.g., opticalgrating) to separate emission beams (e.g., fluorescent beams) from thevarious fluorophores. Additionally, or alternately, the excitation beamscan be impinged on the sample one at a time (e.g., using time-divisionmultiplexing). Additional optical elements and/or employingtime-division multiplexing can increase the complexity of the imagingsystem and/or slow the speed of data acquisition. Subject matterdescribed herein can improve image acquisition speed by using excitationbeams that are spatially separate. This can result in emission beamsthat can be simultaneously detected without employing additional opticsto separate the emission beams and/or detect one emission beam at atime.

In another aspect, there is disclosed a method of imaging a sample. Inembodiments, the sample includes a first fluorophore and a secondfluorophore (e.g., a first fluorophore type and a second fluorophoretype). The sample may contain a plurality of fluorophores of at leastfour different types, or fluorophore types having different absorptionand emission profiles. In embodiments, the sample includes a firstfluorophore, a second fluorophore, a third fluorophore, and a fourthfluorophore, wherein each fluorophore has a different fluorescentemission profile. In embodiments, the method includes directing a firstexcitation beam and second excitation beam onto the sample, wherein theinteraction of the first excitation beam with the first fluorophoregenerates a first fluorescent emission, and the interaction of thesecond excitation beam with the second fluorophore generates a secondfluorescent emission. In embodiments, the method includes directing afirst excitation beam and second excitation beam onto a sample, whereinthe interaction of the first excitation beam with the first fluorophoregenerates a first fluorescent emission, the interaction of the secondexcitation beam with the second fluorophore generates a secondfluorescent emission; the interaction of the first excitation beam witha third fluorophore generates a third fluorescent emission; and theinteraction of the second excitation beam with a fourth fluorophoregenerates a fourth fluorescent emission. In embodiments, the methodincludes directing the first fluorescent emission to the first sensorarray, and the second fluorescent emission to the second sensor array,wherein the first fluorescent emission impinges upon and generates afirst charge (i.e., absorbs at the photodiode) that travels across thefirst sensor array, wherein the second fluorescent emission impingesupon and generates a second charge (i.e., absorbs at the photodiode)that travels across the second sensor array, and wherein the travel ofat least one of the first and the second charge is synchronized with thesample stage speed. In embodiments, the method includes directing thefirst fluorescent emission to the first sensor array, the secondfluorescent emission to the second sensor array, the third fluorescentemission to the third sensor array, the fourth fluorescent emission tothe fourth sensor array, wherein the first fluorescent emission impingesupon and generates a first charge that travels across the first sensorarray, wherein the second fluorescent emission impinges upon andgenerates a second charge that travels across the second sensor array,wherein the third fluorescent emission impinges upon and generates athird charge that travels across the third sensor array, wherein thefourth fluorescent emission impinges upon and generates a fourth chargethat travels across the fourth sensor array, and wherein the travel ofat least one of the first, the second, the third and the fourth chargeis synchronized with the sample stage speed. In embodiments, the speedof all four charges is synchronized with the sample stage speed. Inembodiments, the speed of two charges (e.g., the first and the thirdcharge) is synchronized with the sample stage speed. It is understoodthat when a charge travels across a sensor array (e.g., a first chargethat travels across a first sensor array) that the charge is transferredacross the array according to currently understood theories in the art.For example, the charge transfer originates from the thermal motionphenomenon of electrons, where the electrons which have enough thermalvelocity in the transfer direction will cross the barrier on the chargetransfer path (i.e., across the image sensor). Light energy incident onthe sensor is transformed into an electric signal for digitization,which is transferred to a computing device.

In another aspect, there is disclosed an imaging system including: asample stage moving at a sample stage speed, wherein the sample stage isconfigured to receive a sample including a first fluorophore and asecond fluorophore; a first sensor array and a second sensor array; afirst light source configured to provide a first excitation beam and asecond light source configured to provide a second excitation beam; afirst optical system configured to direct a first excitation beam andsecond excitation beam onto a sample, wherein the interaction of thefirst excitation beam with the first fluorophore generates a firstfluorescent emission, and the interaction of the second excitation beamwith the second fluorophore generates a second fluorescent emission; asecond optical system configured to direct the first fluorescentemission to the first sensor array, and the second fluorescent emissionto the second sensor array, wherein the first fluorescent emissionimpinges upon and generates a first charge that travels across the firstsensor array, wherein the second fluorescent emission impinges upon andgenerates a second charge that travels across the second sensor array;and wherein the travel of at least one of the first and the secondcharge is synchronized with the sample stage speed. In embodiments, theimaging system further includes a third fluorophore, and a fourthfluorophore; a third sensor array, and a fourth sensor array, whereinthe interaction of the first excitation beam with the third fluorophoregenerates a third fluorescent emission, and the interaction of thesecond excitation beam with a fourth fluorophore generates a fourthfluorescent emission; wherein the second optical system is configured todirect the third fluorescent emission to the third sensor array and thefourth fluorescent emission to the fourth sensor array, wherein thethird fluorescent emission impinges upon and generates a third chargethat travels across the third sensor array, wherein the fourthfluorescent emission impinges upon and generates a fourth charge thattravels across the fourth sensor array, wherein the travel of at leastone of the third and the fourth charge is synchronized with the samplestage speed.

In another aspect, there is disclosed an imaging system including: asample stage moving at a sample stage speed, wherein the sample stageincludes a sample including a first fluorophore (e.g., a plurality of afirst type of fluorophore), a second fluorophore (e.g., a plurality of asecond type of fluorophore), a third fluorophore (e.g., a plurality of athird type of fluorophore), and a fourth fluorophore (e.g., a pluralityof a fourth type of fluorophore); a first sensor array, second sensorarray, third sensor array, and a fourth sensor array; a first lightsource configured to provide a first excitation beam and a second lightsource configured to provide a second excitation beam; a first opticalsystem configured to direct a first excitation beam and secondexcitation beam onto a sample, wherein the interaction of the firstexcitation beam with the first fluorophore generates a first fluorescentemission, the interaction of the second excitation beam with the secondfluorophore generates a second fluorescent emission, the interaction ofthe first excitation beam with a third fluorophore generates a thirdfluorescent emission, and the interaction of the second excitation beamwith a fourth fluorophore generates a fourth fluorescent emission; asecond optical system configured to direct the first fluorescentemission to the first sensor array, the second fluorescent emission tothe second sensor array, the third fluorescent emission to the thirdsensor array, the fourth fluorescent emission to the fourth sensorarray, wherein the first fluorescent emission impinges upon andgenerates a first charge that travels across the first sensor array,wherein the second fluorescent emission impinges upon and generates asecond charge that travels across the second sensor array, wherein thethird fluorescent emission impinges upon and generates a third chargethat travels across the third sensor array, wherein the fourthfluorescent emission impinges upon and generates a fourth charge thattravels across the fourth sensor array, wherein the travel of at leastone of the first, the second, the third and the fourth charge issynchronized with the sample stage speed. In embodiments, eachfluorophore type is different. For example, the first fluorophore may bea cyanine dye (e.g., CY3B) and the second fluorophore may be a differentcyanine dye (e.g., Alexa®Fluor 647). In embodiments, each fluorophore(e.g., the first fluorophore, a second fluorophore, a third fluorophore,and a fourth fluorophore) is spectrally distinct. In embodiments, eachfluorophore includes a maximum emission of 405 nm, 470 nm, 488 nm, 514nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912nm, 1024 nm, or 1500 nm.

In embodiments, an aerial image is formed when the fluorescent emissionimpinges upon the sensor array. An aerial image is an image formed bythe emission in the plane of the sensor. In embodiments, the aerialimage, and movement thereof, is synchronized with the sample stage. Inembodiments, the electric charge generated in the sensor by thefluorescent emission travels across the sensor array, wherein thetransfer of charge from one row of the sensor array to the next issynchronized with the stage motion. In embodiments, as an image sweepsover each sensor array (i.e., as the sample is scanned), one or morepixels of the sensor array collect a charge. At certain time intervals,the charge in the pixels in each of the rows of pixels is moved to theiradjacent rows, in the same direction and velocity as the sample scan.Accumulation of charge can integrate during the entire time required forthe row of charge to move from one end of the sensor array to the otherend of the sensor array within the image system.

In embodiments, the first optical system is configured to direct thefirst excitation beam to a first region of the sample at a firstincidence angle, and direct the second excitation beam to a secondregion of the sample at a second incidence angle. In embodiments, thefirst and the second excitation beams are spatially separated andimpinge on the sample at different locations. As a result, fluorescentemissions generated by the first excitation beam are spatially separatedfrom the fluorescent emissions generated by the second excitation beams.For example, the first and third fluorescent emissions generated by thefirst excitation beam are spatially separated from the second and thefourth fluorescent emissions generated by the second excitation beams.

In embodiments, the first optical system is configured to direct thefirst excitation beam to a first region of the sample and direct thesecond excitation beam to a second region of the sample, wherein thefirst region and second region are separated by about 10 μm to about 500μm. In embodiments, the first region and second region are separated byabout 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. Inembodiments, the first region and second region are separated by about 1μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7μm, about 8 μm, about 9 μm, or about 10 μm. In embodiments, the firstregion and second region are separated by about 30 μm, about 35 μm,about 40 μm, about 45 μm, about 50 μm, about 55 μm, or about 60 μm. Inembodiments, the first region and second region are separated by about50 μm. In embodiments, the first region and second region are separatedby about 50 μm, about 51 μm, about 52 μm, about 53 μm, about 54 μm,about 55 μm, about 56 μm, about 57 μm, about 58 μm, about 59 μm, orabout 60 μm.

In embodiments the emission, (e.g., a beam of light or a plurality ofphotons) forms an image in a particular location in space, e.g., in theplane of the image sensor. In embodiments, the emission beam absorbs onthe image sensor and an image formed by this emission in the plane ofthe sensor (i.e., an aerial image, to use a term from lithography) issaid to travel across the sensor while the sample (the object that givesrise to the image) is traveling on the sample stage.

In embodiments, the second optical system includes a first opticalelement including: a first surface configured to reflect the firstfluorescent emission towards the first sensor array, and reflect thesecond fluorescent emission towards the second sensor array; and asecond surface configured to reflect the third fluorescent emissiontowards the third sensor array, and reflect the fourth fluorescentemission towards the fourth sensor array. By reflecting the first andthe third fluorescent emission (generated by the first excitation beam)from the first and the second surface, respectively, spatial separationbetween the first and the third fluorescent emission can be achieved. Byreflecting the second and the fourth fluorescent emission (generated bythe second excitation beam) from the first and the second surface,respectively, spatial separation between the second and the fourthfluorescent emission can be achieved.

In embodiments, the second optical system includes a second opticalelement downstream from the first optical element and is configured tofocus the first fluorescent emission, the second fluorescent emission,the third fluorescent emission, and the fourth fluorescent emission.

In embodiments, the second optical system includes a band pass filterconfigured to selectively transmit the first fluorescent emission, thesecond fluorescent emission, the third fluorescent emission, and thefourth fluorescent emission. A band pass filter selectively passes lightin a wavelength range defined by a center wavelength of maximumradiation transmission (T_(max)) and a bandwidth and blocks passage oflight outside of this range. T_(max) defines the percentage of radiationtransmitted at the center wavelength. In embodiments, the band passfilter is configured to transmit excitation beams having a wavelength of405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm,640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

In embodiments, the first optical element is a dichroic wedge. Inembodiments, the first optical element is a dichroic mirror. Inembodiments, the first optical element includes a dichroic wedge. Inembodiments, the first optical element is a dichroic filter.

In embodiments, a detection camera includes the first sensor array, thesecond sensor array, the third sensor array, and the fourth sensorarray. In embodiments, the detection camera includes the first sensorarray and the second sensor array. In embodiments, the detection cameraincludes the third sensor array and the fourth sensor array. Inembodiments, the imaging system includes one camera. In embodiments, theimaging system includes two cameras. In embodiments, the imaging systemincludes three cameras. In embodiments, the imaging system includes fourcameras. In embodiments, the camera includes 1 image sensor. Inembodiments, the camera includes 2 image sensors. In embodiments, thecamera includes 3 image sensors. In embodiments, the camera includes 4image sensors. In embodiments, each camera includes one or more imagesensors. In embodiments, the camera includes two image sensors. Inembodiments, the imaging system includes two cameras, wherein eachcamera independently includes two image sensors.

In embodiments, the second optical system includes a first opticalelement configured to reflect the first fluorescent emission towards thefirst sensor array, and transmit the third fluorescent emission towardsthe third sensor array (this provides spatial separation between thefirst and the third fluorescent emission); and reflect the secondfluorescent emission towards the second sensor array, and transmit thefourth fluorescent emission towards the fourth sensor array. Byconfiguring the first optical element to reflect the first fluorescentemission and transmit the third fluorescent emission, spatial separationbetween the first and the third fluorescent emission is achieved. Byconfiguring the second optical element to reflect the second fluorescentemission and transmit the fourth fluorescent emission, spatialseparation between the second and the fourth fluorescent emission isachieved. In embodiments, the imaging system spatially separates theexcitation and emission beams.

In embodiments, the second optical system includes: a first lensdownstream from the first optical element and configured to focus thefirst fluorescent emission and the third fluorescent emission; and asecond lens downstream from the first optical element and configured tofocus the second fluorescent emission and the fourth fluorescentemission.

In embodiments, the second optical system includes: a first band passfilter configured to selectively transmit the first fluorescent emissionand the third fluorescent emission; and a second band pass filterconfigured to selectively transmit the second fluorescent emission andthe fourth fluorescent emission.

In embodiments, a first detection camera includes the first sensor arrayand the third sensor array, and a second detection camera includes thesecond sensor array and the fourth sensor array.

In embodiments, the first optical element is an optical filter. Inembodiments, the first optical element is a dichroic beamsplitter.

In embodiments, each sensor array is a TDI sensor array. A sensor arrayrefers to a device or apparatus having a plurality of elements thatconvert the energy of contacted photons into an electrical response. Theterm “time delay integration” or “TDI” refers to sequential detection ofdifferent portions of a sample by different subsets of elements of adetector array, wherein transfer of charge between the subsets ofelements proceeds at a rate synchronized with and in the same directionas the apparent motion of the sample being imaged. For example, TDI canbe carried out by scanning a sample such that a frame transfer deviceproduces a continuous video image of the sample by means of a stack oflinear arrays aligned with and synchronized to the apparent movement ofthe sample, whereby as the image moves from one line to the next, thestored charge moves along with it. Accumulation of charge can integrateduring the entire time required for the row of charge to move from oneend of the sensor array to the other end of the sensor array.

In embodiments, the sensor array (e.g., TDI sensor array) can beconfigured for binning. Binning increases the detector array'ssensitivity by summing the charges from multiple pixels in the arrayinto one pixel. Exemplary types of binning that can be used includehorizontal binning, vertical binning, or full binning. With horizontalbinning, pairs of adjacent pixels in each line of a detector array aresummed. With vertical binning, pairs of adjacent pixels from two linesin the array are summed. Full binning is a combination of horizontal andvertical binning in which four adjacent pixels are summed. For example,binning may include horizontal (1×2), vertical (2×1), or combined (2×2).In embodiments, the sensor array (e.g., TDI sensor array) is notconfigured for binning.

In embodiments, a first subset of sensors of the first sensor array areactivated when the first cross-section overlaps with the first subset ofsensors, and a second subset of sensors of the first sensor array areactivated when the first cross-section overlaps with the second subsetof sensors. In embodiments, a third subset of sensors of the secondsensor array are activated when the second cross-section overlaps withthe third subset of sensors, and a fourth subset of sensors of thesecond sensor array are activated when the second cross-section overlapswith the fourth subset of sensors. In embodiments, a first timedifference between the activation of the first subset of sensors and theactivation of the second subset of sensors of the first sensor array isbased on a first speed of travel of the first cross-section andseparation between the first subset of sensors and the second subset ofsensors. In embodiments, a second time difference between the activationof the third subset of sensors and the activation of the fourth subsetof sensors of the second sensor array is based on a second speed oftravel of the second cross-section and separation between the thirdsubset of sensors and the fourth subset of sensors.

In embodiments, the speed of the sample stage moves at a rate of about 1mm/second to about 50 mm/second. In embodiments, the speed of the samplestage moves at a rate of about 10 mm/second to about 30 mm/second. Inembodiments, the speed of the sample stage moves at a rate of about 15mm/second to about 25 mm/second. In embodiments, the speed of the samplestage moves at a rate of about 20 mm/second. The sample stage isconfigured to receive or support a sample, for example a samplecomprising a flow cell, reaction vessel, or other substrate wherein theflow cell, reaction vessel, or other substrate includes one or moreobjects to be imaged (e.g., biomolecules). The sample stage isconfigured to move along any of x/y/z axes, which are oriented and/oraligned relative to the sample stage. In embodiments, the sample stageincludes a precision mounting plate. A precision mounting plate may befabricated with alignment surfaces, such as mounting pins, grooves,slots, grommets, tabs, magnets, datum surfaces, tooling balls, or othersurfaces designed to accept subassemblies or modules of interest.

In embodiments, each sensor array is at least 2,000 pixels wide. Inembodiments, each sensor array is at least 4,000 pixels wide. Inembodiments, each sensor array is at least 8,000 pixels wide. Inembodiments, each sensor array is at least 12,000 pixels wide. Inembodiments, each sensor array is at least 16,000 pixels wide. Inembodiments, each sensor array is at least 16 pixels long. Inembodiments, each sensor array is at least 32 pixels long. Inembodiments, each sensor array is at least 64 pixels long. Inembodiments, each sensor array is at least 128 pixels long. Inembodiments, each sensor array is at least 256 pixels long. Inembodiments, each sensor array is at least 8,000 pixels wide and atleast 64 pixels long. In embodiments, each sensor array is at least8,000 pixels wide and at least 128 pixels long. In embodiments, eachsensor array is at least 8,000 pixels wide and at least 256 pixels long.In embodiments, each sensory array is rectangular (i.e., two sides ofthe equiangular quadrilateral is longer than the other two sides). Inembodiments, each sensory array is square (i.e., all four sides of theequiangular quadrilateral are equal).

In embodiments, each sensor array is about 1,000 pixels wide to about20,000 pixels wide. In embodiments, each sensor array is about 3,000pixels wide to about 10,000 pixels wide. In embodiments, each sensorarray is about 5,000 pixels wide to about 9,000 pixels wide. Inembodiments, each sensor array is about 1,000 pixels wide. Inembodiments, each sensor array is about 2,000 pixels wide. Inembodiments, each sensor array is about 3,000 pixels wide. Inembodiments, each sensor array is about 4,000 pixels wide. Inembodiments, each sensor array is about 5,000 pixels wide. Inembodiments, each sensor array is about 6,000 pixels wide. Inembodiments, each sensor array is about 7,000 pixels wide. Inembodiments, each sensor array is about 8,000 pixels wide. Inembodiments, each sensor array is about 9,000 pixels wide. Inembodiments, each sensor array is about 10,000 pixels wide. Inembodiments, each sensor array is about 11,000 pixels wide. Inembodiments, each sensor array is about 12,000 pixels wide. Inembodiments, each sensor array is about 13,000 pixels wide. Inembodiments, each sensor array is about 2,000 pixels wide. Inembodiments, each sensor array is about 4,000 pixels wide. Inembodiments, each sensor array is about 8,000 pixels wide. Inembodiments, each sensor array is about 12,000 pixels wide. Inembodiments, each sensor array is about 16,000 pixels wide. Inembodiments, each sensor array is about 16 pixels long. In embodiments,each sensor array is about 32 pixels long. In embodiments, each sensorarray is about 64 pixels long. In embodiments, each sensor array isabout 128 pixels long. In embodiments, each sensor array is about 256pixels long. In embodiments, each sensor array is about 8,000 pixelswide and about 64 pixels long. In embodiments, each sensor array isabout 8,000 pixels wide and about 128 pixels long. In embodiments, eachsensor array is about 8,000 pixels wide and about 256 pixels long. Inembodiments, each sensor array is about 32 pixels long. In embodiments,each sensor array is about 64 pixels long. In embodiments, each sensorarray is about 256 pixels long. In embodiments, each sensor array isabout 512 pixels long. In embodiments, each sensor array is about 10 to300 pixels long. In embodiments, each sensor array is about 32 to about256 pixels long. In embodiments, each sensor array is about 32 to about64 pixels long.

In embodiments, the sample stage is a motorized translation stage. Inembodiments, the motor is a stepper motor, piezo motor, brushless motor,hysteresis motor, linear motor, or a servomotor. In embodiments, themotor is a stepper motor. In embodiments, the stepper motor includes anintegrated ball spline. In embodiments, the motor is a piezo motor. Inembodiments, the motor is a brushless motor. In embodiments, the motoris a hysteresis motor. In embodiments, the motor is a linear motor. Inembodiments, the motor is a servomotor. In embodiments, the servomotorincludes a braking mechanism. In embodiments, the motor is a Picomotor™actuator.

In embodiments, the sample stage is configured to receive and retain asample. In embodiments, the sample stage is configured to receive andretain a reaction vessel containing a sample (e.g., a flow cell asdescribed herein). In embodiments, the sample stage includes a positionencoder, wherein the position encoder generates a synchronization signalthat synchronizes the travel of the fluorescent emissions. Inembodiments, the imaging system further includes an absolute encoder. Anabsolute encoder provides information about the position (i.e., thedistance) the camera, the image sensor, and/or the lens, relative to thesample stage and/or the sample. The absolute position encoder not onlyprovides highly repeatable positioning, but also enables the recovery ofa previously-saved position if rescanning a previously imaged region.

In embodiments, the sample stage includes, and optionally retains, areaction vessel, flow cell, substrate, or multiwell container. Thoseskilled in the art will recognize that a flow cell or other supportstructure may be used with any of a variety of arrays known in the artto achieve similar results. Such arrays may be formed by arrangingbiological components of samples randomly or in predefined patterns onthe surfaces of the support by any known technique. The term “multiwellcontainer” as used herein, refers to a substrate comprising a surface,the surface including a plurality of reaction chambers separated fromeach other by interstitial regions on the surface. In embodiments, themicroplate has dimensions as provided and described by American NationalStandards Institute (ANSI) and Society for Laboratory Automation AndScreening (SLAS); for example the tolerances and dimensions set forth inANSI SLAS 1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004(R2012); ANSI SLAS 4-2004 (R2012); and ANSI SLAS 6-2012, which areincorporated herein by reference. The dimensions of the microplate asdescribed herein and the arrangement of the reaction chambers may becompatible with an established format for automated laboratoryequipment.

The reaction chambers may be provided as wells (alternatively referredto as reaction chambers), for example a multiwell container may contain2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells. In embodiments, the96 and 384 wells are arranged in a 2:3 rectangular matrix. Inembodiments, the 24 wells are arranged in a 3:8 rectangular matrix. Inembodiments, the 48 wells are arranged in a 3:4 rectangular matrix. Inembodiments, the reaction chamber is a microscope slide (e.g., a glassslide about 75 mm by about 25 mm). In embodiments the slide is aconcavity slide (e.g., the slide includes a depression). In embodiments,the slide includes a coating for enhanced biomolecule adhesion (e.g.,poly-L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, orgold). In embodiments, the multiwell container is about 5 inches byabout 3.33 inches, and includes a plurality of 5 mm diameter wells. Inembodiments, the multiwell container is about 5 inches by about 3.33inches, and includes a plurality of 6 mm diameter wells. In embodiments,the multiwell container is about 5 inches by about 3.33 inches, andincludes a plurality of 7 mm diameter wells. In embodiments, themultiwell container is about 5 inches by about 3.33 inches, and includesa plurality of 7.5 mm diameter wells. In embodiments, the multiwellcontainer is 5 inches by 3.33 inches, and includes a plurality of 7.5 mmdiameter wells. In embodiments, the multiwell container is about 5inches by about 3.33 inches, and includes a plurality of 8 mm diameterwells. In embodiments, the multiwell container is a flat glass orplastic tray in which an array of wells are formed, wherein each wellcan hold between from a few microliters to hundreds of microliters offluid reagents and samples.

The term “well” refers to a discrete concave feature in a substratehaving a surface opening that is completely surrounded by interstitialregion(s) of the surface. Wells can have any of a variety of shapes attheir opening in a surface including but not limited to round,elliptical, square, polygonal, or star shaped (i.e., star shaped withany number of vertices). The cross section of a well taken orthogonallywith the surface may be curved, square, polygonal, hyperbolic, conical,or angular. The wells of a multiwell container are available indifferent shapes, for example F-Bottom: flat bottom; C-Bottom: bottomwith minimal rounded edges; V-Bottom: V-shaped bottom; or U-Bottom:U-shaped bottom. In embodiments, the well is substantially square. Inembodiments, the well is square. In embodiments, the well is F-bottom.In embodiments, the multiwell container includes 24 substantially roundflat bottom wells. In embodiments, the multiwell container includes 48substantially round flat bottom wells. In embodiments, the multiwellcontainer includes 96 substantially round flat bottom wells. Inembodiments, the multiwell container includes 384 substantially squareflat bottom wells.

The discrete regions (i.e., features, wells) of the multiwell containermay have defined locations in a regular array, which may correspond to arectilinear pattern, circular pattern, hexagonal pattern, or the like.In embodiments, the pattern of wells includes concentric circles ofregions, spiral patterns, rectilinear patterns, hexagonal patterns, andthe like. In embodiments, the pattern of wells is arranged in arectilinear or hexagonal pattern A regular array of such regions isadvantageous for detection and data analysis of signals collected fromthe arrays during an analysis. These discrete regions are separated byinterstitial regions. As used herein, the term “interstitial region”refers to an area in a substrate or on a surface that separates otherareas of the substrate or surface. For example, an interstitial regioncan separate one concave feature of an array from another concavefeature of the array. The two regions that are separated from each othercan be discrete, lacking contact with each other. In another example, aninterstitial region can separate a first portion of a feature from asecond portion of a feature. In embodiments the interstitial region iscontinuous whereas the features are discrete, for example, as is thecase for an array of wells in an otherwise continuous surface. Theseparation provided by an interstitial region can be partial or fullseparation. In embodiments, interstitial regions have a surface materialthat differs from the surface material of the wells (e.g., theinterstitial region contains a photoresist and the surface of the wellis glass). In embodiments, interstitial regions have a surface materialthat is the same as the surface material of the wells (e.g., both thesurface of the interstitial region and the surface of well contain apolymer or copolymer).

In embodiments, the imaging system further includes one or more of thefollowing: a collimating lens, a beam shaping lens, mirrors, or acylindrical lens.

In embodiments, the imaging system further includes one or more linegenerators. In embodiments, the imaging system further includes two linegenerators. In embodiments, one or more line generators (e.g., 2, 4, 6,8, or 10 lines) are used to illuminate the sample. The one or more linegenerators may be configured to produce an excitation line having ashape at the sample that is rectangular or oblong. Exemplary shapesinclude, but are not limited to, a rectangular, elliptical, or ovalshape. In embodiments, one or more excitation lines contacts the sampleto illuminate and/or excite one or more biomolecules in the sample. Inembodiments, the line is rectangular having a height and width. Inembodiments, the height of each line is approximately 1, 2, 3, 4, or 5mm. In embodiments, the height of each line is approximately 1.1 mm, 1.2mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2.0 mm.In embodiments, the width of each line is about 5 μm, 10 μm, 15 μm, 20μm, 25 μm, or about 30 μm. In embodiments, the width of each line is 1.5mm to about 2.0 mm and the height of each line is about 10 μm to about30 μm. In embodiments, the width of each line is 1.6 mm to about 1.7 mmand the height of each line is about 15 μm to about 25 μm. Inembodiments, the width of each line is 1.7 mm and the height of eachline is about 20 μm.

In embodiments, the imaging system is within a microfluidic device. Inan aspect is provided a microfluidic device, wherein the microfluidicdevice includes an imaging system as described herein. In embodiments,the microfluidic device includes one or more reaction vessels or solidsupport where reagents interact and are imaged. Exemplary systems havingfluidic components that can be readily modified for use in a systemherein include, but are not limited to, those set forth in U.S. Pat.Nos. 8,241,573, 8,039,817; or US Pat. App. Pub. No. 2012/0270305 A1,each of which is incorporated herein by reference. In embodiments, themicrofluidic device further includes one or more excitation lasers. Inembodiments, the imaging system is within a bioanalytical instrument. Inembodiments, the bioanalytical instrument further includes a lightsource and an integrated fluidic system of one or more interconnectedchambers, ports, and channels in fluid communication and configured forcarrying out an analytical reaction or processes. In embodiments, thedevice as described herein detects scattered light from the sample. Inembodiments, the device as described herein detects diffracted lightfrom the sample. In embodiments, the device as described herein detectsreflected light from the sample. In embodiments, the device as describedherein detects absorbed light from the sample. In embodiments, thedevice as described herein detects refracted light from the sample. Inembodiments, the device as described herein detects transmitted lightnot absorbed by the sample. In embodiments, the device further includesat least one reservoir physically coupled to the structure. Inembodiments, the reservoir is configured to store one or more reagentsor hold waste material. In some embodiments, the reagents include fluidssuch as water, buffer solution (e.g., an imaging buffer includingascorbic acid), target capture reagents, or nucleic acid amplificationreagents. In some embodiments, the reagent container compartments may beconfigured to maintain the contents of such containers at prescribedstorage temperatures and/or to agitate such containers to maintain thecontents of the containers in solution or suspension. In embodiments,the at least one reservoir includes reaction reagents, for examplenucleic acid amplification reagents (e.g., polymerase and nucleotidesneeded for amplification), and/or nucleic acid sequencing reagents. Inembodiments, the at least one reservoir includes at least one of a wastereservoir, a sequencing reagent reservoir, a clustering reagentreservoir, and a wash solution reservoir. In embodiments, the deviceincludes a plurality of a sequencing reagent reservoirs and clusteringreagent reservoirs. In embodiments, the clustering reagent reservoirincludes amplification reagents (e.g., an aqueous buffer containingenzymes, salts, and nucleotides, denaturants, crowding agents, etc.).

In embodiments, the imaging system may generate image data, for example,at a resolution between 0.1 and 50 microns, which is then forwarded to acontrol/processing system within the bioanalytical instrument. Thecontrol/processing system may perform various operations, such asanalog-to-digital conversion, scaling, filtering, and association of thedata in multiple frames to appropriately and accurately image multiplesites at specific locations on a sample. The control/processing systemmay store the image data and may ultimately forward the image data to apost-processing system where the data is further analyzed. For example,further analysis may include determining nucleotide sequence informationfrom the image data. In embodiments, the control/processing system mayinclude hardware, firmware, and software designed to control operationof the bioanalytical instrument. The image data may be analyzed by thebioanalytical instrument itself, or may be stored for analysis by othersystems and at different times subsequent to imaging. In embodiments,the cameras include an objective lens having high numerical aperture(NA) values. Image data obtained by the optical assembly may have aresolution that is between 0.1 and 50 microns or, more particularly,between 0.1 and 10 microns. In embodiments, the numerical aperture forthe camera is at least 0.2. In embodiments, the numerical aperture forthe camera is no greater than 0.8. In embodiments, the numericalaperture for the camera is no greater than 0.5. Image systems describedherein may have a resolution that is sufficient to individually resolvethe features or sites that are separated by a distance of less than 10μm, 5 μm, 2 μm, 1.5 μm, 1.0 μm, 0.8 μm, 0.5 μm, or less. In embodiments,the image systems described herein may have a resolution that issufficient to individually resolve the features or sites that areseparated by a distance of 100 μm at most. Depending on the sample, forexample the microwells or nanowells of a multiwell container, theimaging system described herein may be configured for wide-fielddetection. The field diameter for the imaging system may be, forexample, at least 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm or larger. Bychoosing suitable optical components, the field diameter can be limitedto a maximum area as well and, as such the field diameter can be, forexample, no larger than 5 mm, 4 mm, 3 mm, 2 mm or 1 mm. For example, inembodiments an image obtained by an imaging system can have an area thatis in a range of about 0.25 mm² to about 25 mm².

In another aspect, there is disclosed a method of imaging a sampleincluding: a) directing a first excitation beam and a second excitationbeam onto a sample, wherein the sample is on a sample stage moving at asample stage speed, wherein the sample comprises a first fluorophorethat generates a first fluorescent emission and a second fluorophorethat generates a second fluorescent emission following interaction witha first excitation beam and a second excitation beam, respectively; b)directing the first fluorescent emission to impinge upon and generate afirst charge that travels across a first sensor array at a first chargespeed, and directing the second fluorescent emission to impinge upon andgenerate a second charge that travels across a second sensor array at asecond charge speed, wherein at least one of the first emission speedand the second emission speed is synchronized with the sample stagespeed; and c) scanning the sample in a scan dimension and repeating stepa) and step b) to form an image of the sample. In embodiments, thesample further includes a third fluorophore that generates a thirdfluorescent emission and a fourth fluorophore that generates a fourthfluorescent emission following interaction with a first excitation beamand a second excitation beam, respectively; and directing said thirdfluorescent emission to impinge upon and generate a third charge thattravels across a third sensor array at a third charge speed, anddirecting said fourth fluorescent emission to impinge upon and generatesa fourth charge that travels across a fourth sensor array at a fourthcharge speed. In embodiments the collective sum of charge within eachpixel travels across the sensor array.

The sample stage is configured to move along any of x/y/z axes, whichare oriented and/or aligned relative to the sample stage. To establish astandard coordinate system and frame of reference, it is useful toprovide a description of the axes. In conventional descriptions ofthree-dimensional space using Cartesian coordinates, there are sixdegrees of freedom. Each degree of freedom corresponds to thetranslation along and rotations around three perpendicular X-, Y-, andZ-axes. A first degree of freedom can be defined as moving left andright along the X-axis. A second degree of freedom can be defined asmoving backward and forward along the Y-axis. A third degree of freedomcan be defined as moving up and down along the Z-axis. A fourth degreeof freedom can be defined as rotating around the X-axis, or “roll” axis,alternatively referred to as the longitudinal axis. A fifth degree offreedom can be defined as rotating around the Y-axis, or “pitch” axis,alternatively referred to as the transverse axis. Used interchangeablythroughout, pitch and roll may be referred to as tip and tilt. A sixthdegree of freedom can be defined as rotating around the Z-axis, or“yaw.” A plane refers to a 2-dimensional (2D) area defined by two axes(e.g., x and y together form the xy plane). When used in reference to adetecting apparatus (e.g., an image sensor) and an object observed bythe detector (e.g., the sample), the xy plane may be specified as beingorthogonal to the direction of observation between the detector andobject being detected. The image plane is a projection of the image on atwo-dimensional plane. For example, in embodiments, the image plane isthe projection of an image on the surface of the image sensor. Inembodiments, the scan axis is the x axis. In embodiments, the scan axisis the y axis.

In embodiments, the travel of the first emission and the travel of thesecond emission speed is synchronized with the sample stage speed.Synchronizing the sample stage with the charge travel across the arrayallows for high precision, and more accurate imaging (e.g., relative toa control, such as not synchronizing the sample stage). Given theplethora of different biomolecules and components within a sample thatmodulate viscosity and thermal sensitivity, controlling the flow rate ofthe sample is not preferred due to the challenges associated withaccurately controlling the rate and maintaining synchrony.

In embodiments, the method includes illuminating a sample and detectinglight from the sample (e.g., fluorescent excitation events, scatteredlight, transmitted light, or reflected light) using the imaging systemdescribed herein. In embodiments, the method includes scanning thesample (i.e., translating the sample relative to the camera). Inembodiments, the method includes illuminating a sample to generatefluorescent events, and detecting one or more fluorescent events usingthe imaging system described herein. In embodiments, the method includesdetecting clusters (e.g., amplified colonies of nucleic acids) on asolid support. In embodiments, the method includes detectingfluorescently labeled nucleotides incorporated into a template nucleicacid. In embodiments, the method includes sequencing one or more nucleicacid templates. In embodiments, the method includes amplifying one ormore nucleic acid templates on a solid support, thereby generating acluster or colony of a plurality of nucleic acid templates at a feature.In embodiments, the method includes incorporating a labeled nucleotideinto a primer hybridized to one or more of the nucleic acid templatesand detecting the incorporated nucleotide. In embodiments, detectingincludes imaging the feature using the imaging system described herein.In embodiments, the sample includes one or more biomolecules. A varietyof biomolecules may be present in the sample. Exemplary biomoleculesinclude, without limitation, nucleic acids such as DNA or RNA, proteinssuch as enzymes or receptors, polypeptides, nucleotides, amino acids,saccharides, cofactors, metabolites or derivatives of these naturalcomponents. Although the systems and methods as described herein arewith respect to biomolecules, it will be understood that other samplesor components can be used as well. For example, synthetic samples can beused such as combinatorial libraries, or libraries of compounds havingspecies known or suspected of having a desired structure or function. Inembodiments, the sample includes one or more fluorescent labels. Inembodiments, the sample includes one or more fluorescently labeledbiomolecules.

In embodiments, the method includes imaging the sample including fourdifferent fluorophores simultaneously. That is, collecting fluorescentemission information for four different fluorophores at the same time.

In embodiments, the first excitation beam and the second excitation beaminclude an excitation line (e.g., an excitation beam provided by a linegenerator). In embodiments, one or more line generators (e.g., 2, 4, 6,8, or 10 lines) are used to illuminate the sample. The one or more linegenerators may be configured to produce an excitation line having ashape at the sample that is rectangular or oblong. Exemplary shapesinclude, but are not limited to, a rectangular, elliptical, or ovalshape. In embodiments, one or more excitation lines contacts the sampleto illuminate and/or excite one or more biomolecules in the sample.

In embodiments, scanning the sample includes moving the sample stage. Inembodiments, scanning the sample includes moving the sample stage at aconstant speed. Movement of the sample stage can be in one or moredimensions including, for example, one or both of the dimensions thatare orthogonal to the direction of propagation for the fluorescentemission and typically denoted as the x and y dimensions. The system mayfurther include a scanning element, which may be a mechanical,electro-mechanical component, software component, or combination thereofconfigured to scan the sample along a direction, which may correspond toa scan direction. In an embodiment, the scan direction is orthogonal tothe excitation direction of the sample. In an embodiment, the scandirection is non-orthogonal to the excitation beam direction, whereinthe orthogonal projected component directly contributes to the finalimage reconstruction. The term “scanning element” is intended to mean anelement capable of sequentially detecting different portions of asample. A scanning element can operate, by changing the position of oneor more component of the system including, for example, the light sourcethe objective lens, the image sensor, or the sample. Exemplary scanningelements include, but are not limited to a galvanometer configured tomove a beam (e.g., excitation beam) across a sample or a translationstage configured to move the sample across the beam. In embodiments, thesample is scanned at about 1 mm²/sec, 1.5 mm²/sec, 5 mm²/sec, 10mm²/sec, 50 mm²/sec or 100 mm²/sec. In embodiments, the sample isscanned at 10 mm²/sec, 20 mm²/sec, 30 mm²/sec, 40 mm²/sec, or 50mm²/sec. In embodiments, the sample is scanned at about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about10 mm²/sec. In embodiments, the sample is scanned at about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19, orabout 20 mm²/sec. In embodiments, the sample is scanned at least 20mm²/sec. In embodiments, the camera is adjusted dynamically as thesample is scanned (e.g., continuously scanned in a scan axis, such asthe x axis). In embodiments, the camera is adjusted initially (e.g.,during a configuration or first cycle of a series of cyclic experiments)as the sample is scanned (e.g., continuously scanned in a scan axis,such as the x axis). For example, if scanning the sample stage over aseries of imaging cycles (e.g., sequencing cycles), an initialconfiguration scan of the sample adjusts the camera to maximize thefocus of the sample and the orientation and/or position of the cameraremains static for remaining imaging cycles.

In embodiments, the method further includes storing a datarepresentation of the image of the sample in a computer readable memory.

In embodiments, the excitation beam includes UV radiation, VISradiation, or IR radiation. In embodiments, the excitation beam includesexcitation beams having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm,520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm,1024 nm, or 1500 nm.

In embodiments, the illuminator or light source is a radiation source(e.g., an origin or generator of propagated electromagnetic energy)providing incident light to the sample. A radiation source can includean illumination source producing electromagnetic radiation in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 390to 770 nm), or infrared (IR) range (about 0.77 to 25 microns), or otherrange of the electromagnetic spectrum. In embodiments, the illuminatoror light source is a lamp such as an arc lamp or quartz halogen lamp. Inembodiments, the illuminator or light source is a coherent light source.In embodiments, the light source is a laser, LED (light emitting diode),a mercury or tungsten lamp, or a super-continuous diode. In embodiments,the light source provides excitation beams having a wavelength between200 nm to 1500 nm. In embodiments, the laser provides excitation beamshaving a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm,561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500nm. In embodiments, the laser provides excitation beams having awavelength of 405 nm, 488 nm, 532 nm, or 633 nm.

In embodiments, the light source provides one or more excitation beams.An excitation beam is intended to mean electromagnetic energy propagatedtoward a sample or sample region. An excitation beam may be shaped suchthat the collection of electromagnetic waves or particles are propagatedin a uniform direction, wherein the 2-dimensional cross sectionorthogonal to the direction of propagation is rectangular or oblong.Exemplary 2-dimensional cross sections of an excitation beam can includea rectangular, elliptical, or oval shape. The cross-sectional width ofan excitation beam can have one or both dimensions in a range of, forexample, about 0.5 μm to about 50 μm. For example, a dimension of theexcitation beam can be at least about 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5μm or 10 μm. Furthermore, a dimension of a excitation beam can be, forexample, at most about 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 5 μm or 10 μm. Inembodiments, a dimension of a excitation beam is about 0.2 μm to about50 μm. In embodiments, a dimension of a excitation beam is 10 μm toabout 30 μm. In embodiments, a dimension of a excitation beam is 20 μmto about 30 μm. In embodiments, a dimension of a excitation beam is 20μm. It will be understood that these dimensions are merely exemplary andexcitation beams having other dimensions can be used if desired.

In embodiments, the light source is a laser (e.g., a laser such as asolid-state laser or a gas laser). In embodiments, the light sourceincludes one or more vertical cavity surface emitting lasers (VCSELs),vertical external cavity surface emitting lasers (VECSELs), or diodepumped solid state (DPSS) lasers. In embodiments, the light source is acontinuous wave (CW) laser or a pulsed laser. In embodiments, the lightsource is a pulsed laser. In embodiments, the light source is anultrashort pulsed laser. An ultrashort laser is a laser capable ofproducing excitation beams for a time duration of a picosecond or less.An ultrashort laser typically includes additional components, such as apulse controller, pulse shaper, and spatial light modulator, and thelike for controlling the pulse of excitation beams. In embodiments, theultrashort laser provides excitation beams for femtoseconds orpicoseconds. In embodiments, the light source is a pulsed femtosecond orpicosecond laser. In embodiments, the laser is a Ti-sapphire laser, adye-laser, or a fiber laser. In embodiments, the system includes two ormore light sources (e.g., lasers). In embodiments, the first lightsource configured to emit light in red wavelengths, and a second lightsource configured to emit light in green wavelengths. In embodiments,the system includes two or more lasers.

In embodiments, the sample includes modified nucleotides (e.g.,nucleotides including a reversible terminator and/or a label). Inembodiments, the sample includes nucleotides attached to a substrate. Inembodiments, the sample includes surface-immobilized polynucleotides(e.g., polynucleotide primers or polynucleotide templates that arecovalently attached to the substrate). In embodiments, the 5′ end of thepolynucleotides contains a functional group that is tethered to thesolid support. Non-limiting examples of covalent attachment includeamine-modified polynucleotides reacting with epoxy or isothiocyanategroups on the solid support, succinylated polynucleotides reacting withaminophenyl or aminopropyl functional groups on the solid support,dibenzocycloctyne-modified polynucleotides reacting with azidefunctional groups on the solid support (or vice versa),trans-cyclooctyne-modified polynucleotides reacting with tetrazine ormethyl tetrazine groups on the solid support (or vice versa), disulfidemodified polynucleotides reacting with mercapto-functional groups on thesolid support, amine-functionalized polynucleotides reacting withcarboxylic acid groups on the core via1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)chemistry, thiol-modified polynucleotides attaching to a solid supportvia a disulphide bond or maleimide linkage, alkyne-modifiedpolynucleotides attaching to a solid support via copper-catalyzed clickreactions to azide functional groups on the solid support, andacrydite-modified polynucleotides polymerizing with free acrylic acidmonomers on the solid support to form polyacrylamide or reacting withthiol groups on the solid support.

In embodiments, the substrate is glass or quartz, such as a microscopeslide, having a surface that is uniformly silanized. This may beaccomplished using conventional protocols e.g., Beattie et al (1995),Molecular Biotechnology, 4: 213. Such a surface is readily treated topermit end-attachment of oligonucleotides (e.g., forward and reverseprimers, and/or a splint primer) prior to amplification. In embodimentsthe solid support surface further includes a polymer coating, whichcontains functional groups capable of immobilizing polynucleotides. Insome embodiments, the solid support includes a patterned surfacesuitable for immobilization of polynucleotides in an ordered pattern. Apatterned surface refers to an arrangement of different regions in or onan exposed layer of a solid support. For example, one or more of theregions can be features where one or more primers are present. Thefeatures can be separated by interstitial regions where capture primersare not present. In some embodiments, the pattern can be an x-y formatof features that are in rows and columns. In some embodiments, thepattern can be a repeating arrangement of features and/or interstitialregions. In some embodiments, the pattern can be a random arrangement offeatures and/or interstitial regions. In some embodiments, the primersare randomly distributed upon the solid support. In some embodiments,the primers are distributed on a patterned surface.

In embodiments, the sample includes an array having a plurality ofindividual sites (e.g., a microarray or multiwell container). A typicalmicroarray contains sites, sometimes referred to as features, eachhaving a population of targets. Sites or features of an array aretypically discrete, being separated with spaces between each other. Thesize of the features and the spacing between the features can vary suchthat arrays can be high density, medium density or lower density. Highdensity arrays are characterized as having sites separated by less thanabout 15 μm. Medium density arrays have sites separated by about 15 to30 μm, while low density arrays have sites separated by greater than 30μm. In embodiments, the sample is an array including features that areseparated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm. Otherexemplary samples include, but are not limited to, biological specimens(e.g., a nucleic acid, a protein, a cell, a virus, or a tissue),nanoparticles, or electronic chips (e.g., a microprocessor chip). Asample refers to an object or location that is intended to be detected.In embodiments, a sample includes a plurality of distinct features thatare targets for imaging. In some embodiments a sample includes anon-planar structure with a surface, such as a bead or a well, to whichtarget nucleic acids have been attached as the target features. Inembodiments, the sample is held by a sample holder. The sample holdercan be a multiwell plate. In some instances, the multiwell plate has 16,24, 48, 96, 384 or more sample wells. In some of these instances, anarray of light sources, e.g., LEDs, has 16, 24, 48, 96, 384 or morecorresponding light sources. In some instances, the multiwell plate is astandard microwell plate for biological analysis. In embodiments, thesample holder is coated, at least internally, with a material forpreventing a biological materials from sticking to the sample holder,such as a fluorinated polymer or BSA. In embodiments, the sampleincludes genomic material which may be sequenced. In embodiments, thesample includes labeled nucleotides, for example nucleotides containingdifferent labels corresponding to different wavelengths of light. Thelabels may be, for example, fluorescent, chemiluminescent orbioluminescent labels. For example, in gene sequencing (or DNAsequencing), embodiments may be used to determine the precise order ofnucleotide bases within a nucleic acid polynucleotide (e.g., a strand ofDNA). The nucleotide bases may be labeled with a specific fluorescentlabel (e.g., adenine (A), guanine (G), cytosine (C), or thymine (T)).Alternatively, one color, two color, or three color sequencing methods,for example, may be used. With respect to fluorescence, each of thenucleotide bases may be determined in order by successively exciting thenucleic acid with excitation light. The nucleic acid may absorb theexcitation light and transmit an emitted light of a different wavelengthonto an image sensor as described herein. The image sensor may measurethe wavelength of emitted light and intensity received by thephotodiode. Each nucleotide (e.g., fluorescently labeled nucleotide),when excited by excitation light of a certain wavelength and/orintensity, may emit a certain wavelength of light and/or intensity intothe image sensor, allowing identification of the presence of aparticular nucleotide base at a particular position in the nucleic acid.Once that particular nucleotide base has been determined, it may beremoved from the nucleic acid, such that the next successive nucleotidebase may be determined according to a similar process.

In embodiments, the sample includes a microplate array, including: asubstrate including a surface, the surface comprising a plurality ofwells separated from each other by interstitial regions on the surface,wherein one or more wells includes a sample (e.g., a cell or tissuesample), particle, or nucleic acid. In embodiments, the sample includesa cell. In embodiments, the sample includes a particle. In embodiments,the sample includes a nucleic acid. In embodiments, the sample is atissue sample. In embodiments, the sample includes a cell. Inembodiments, the surface is substantially free of oligonucleotides. Inembodiments, the microplate array includes 2, 4, 6, 12, 24, 48, 96, 384or 1536 wells. In embodiments, the microplate array includes 24, 48, 96,or 384 wells. In embodiments, the microplate array includes 24 wells. Inembodiments, the microplate array includes 48 wells. In embodiments, themicroplate array includes 96 wells. In embodiments, the microplate arrayincludes 384 wells. In embodiments, the dimensions of the microplateconform to the standards provided by the American National StandardsInstitute (ANSI) and Society For Laboratory Automation And Screening(SLAS); for example the tolerances and dimensions set forth in ANSI SLAS1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSISLAS 4-2004 (R2012); and ANSI SLAS 6-2012. In embodiments, themicroplate has a rectangular shape that measures 127.7 mm±0.5 mm inlength by 85.4 mm±0.5 mm in width, and includes 6, 12, 24, 48, or 96wells. In embodiments, the microplate has a rectangular shape thatmeasures 127.7 mm±0.5 mm in length by 85.4 mm±0.5 mm in width, andincludes 6, 12, 24, 48, or 96 wells, wherein each well has an averagediameter of about 5-7 mm. In embodiments, the microplate has arectangular shape that measures 127.7 mm±0.5 mm in length by 85.4 mm±0.5mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each wellhas an average diameter of about 6 mm.

A general overview of an example workflow is provided in FIGS. 1-2 .Described herein is an imaging system that includes at least oneCCD-CMOS sensor array (alternatively referred to herein as a TDI arrayor hybrid TDI line scan sensor). A hybrid TDI sensor (e.g., a CCD-CMOSsensor array) combines the CCD pixel structure with CMOS technologyenabling ultra-high-speed image captures with greater sensitivity,relative to conventional CCD and/or conventional CMOS sensors. Inembodiments, the CCD-CMOS sensor array captures a plurality (e.g., tensof hundreds of lines), wherein each line successively captures asnapshot of the sample as it passes, allowing the accumulation ofmultiple images that can result in a very low noise image of a dark orhard-to-image object.

In embodiments, the sample is an array (e.g., a microarray). A typicalmicroarray contains sites, sometimes referred to as features, eachhaving a population of targets. Sites or features of an array aretypically discrete, being separated with spaces between each other. Thesize of the features and the spacing between the features can vary suchthat arrays can be high density, medium density or lower density. Highdensity arrays are characterized as having sites separated by less thanabout 15 μm. Medium density arrays have sites separated by about 15 to30 μm, while low density arrays have sites separated by greater than 30μm. In embodiments, the sample is an array including features that areseparated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm. Otherexemplary samples include, but are not limited to, biological specimens(e.g., a nucleic acid, a protein, a cell, a virus, or a tissue),nanoparticles, or electronic chips (e.g., a microprocessor chip). Inembodiments, the sample includes a microplate array, including: asubstrate including a surface, the surface comprising a plurality ofwells separated from each other by interstitial regions on the surface,wherein one or more wells includes a sample (e.g., a cell or tissuesample), particle, or nucleic acid. In embodiments, the sample includesa cell. In embodiments, the sample includes a particle. In embodiments,the sample includes a nucleic acid. In embodiments, the sample is atissue sample. In embodiments, the sample includes a cell. Inembodiments, the surface is substantially free of oligonucleotides. Inembodiments, the microplate array includes 2, 4, 6, 12, 24, 48, 96, 384or 1536 wells. In embodiments, the microplate array includes 24, 48, 96,or 384 wells. In embodiments, the microplate array includes 24 wells. Inembodiments, the microplate array includes 48 wells. In embodiments, themicroplate array includes 96 wells. In embodiments, the microplate arrayincludes 384 wells. In embodiments, the dimensions of the microplateconform to the standards provided by the American National StandardsInstitute (ANSI) and Society For Laboratory Automation And Screening(SLAS); for example the tolerances and dimensions set forth in ANSI SLAS1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSISLAS 4-2004 (R2012); and ANSI SLAS 6-2012. In embodiments, themicroplate has a rectangular shape that measures 127.7 mm±0.5 mm inlength by 85.4 mm±0.5 mm in width, and includes 6, 12, 24, 48, or 96wells. In embodiments, the microplate has a rectangular shape thatmeasures 127.7 mm±0.5 mm in length by 85.4 mm±0.5 mm in width, andincludes 6, 12, 24, 48, or 96 wells, wherein each well has an averagediameter of about 5-7 mm. In embodiments, the microplate has arectangular shape that measures 127.7 mm±0.5 mm in length by 85.4 mm±0.5mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each wellhas an average diameter of about 6 mm.

In embodiments, the imaging system includes one camera and one CCD-CMOSsensor array per channel. In embodiments, the imaging system includesone camera and four CCD-CMOS sensors. In embodiments, the imaging systemincludes two cameras and two CCD-CMOS sensor arrays. In embodiments, theimaging system architecture includes an upright infinity-correctedepifluorescence microscope that uses two cameras, each of themresponsible for capturing two of the four color channels. Inembodiments, the imaging path includes a beamsplitter that routes thefour fluorescence bands to a first camera or a second camera. Inembodiments, each of the branches of the imaging path contains a dualbandpass filter designed specifically to transmit the pair of colorchannels intended for that branch, and to block the excitation light andout-of-band fluorescence.

Typical implementations within a multicolor image acquisition relies ontaking two sequential exposures with red and green excitation light tocapture images in multiple channels. In contrast to this, the imagingsystem described herein images all channels in parallel and relies onspatial separation of excitation light, instead of temporal separation,see FIG. 1 and FIG. 2 . An advantage of the systems and methodsdescribed herein is that they provide for rapid and efficient detectionof a plurality of target nucleic acid in parallel.

In a first configuration workflow, as shown in FIG. 1 , a first lightsource 101 provides a first excitation beam 103 and a second lightsource 102 provides a second excitation beam 104. Excitation beam 103 isdirected by mirror 112, mirror 113, and lens 110 towards dichroic filter140. Excitation beam 104 is directed by mirror 111, mirror 113, and lens110 towards dichroic filter 140. Dichroic filter 140 directs excitationbeam 103 and excitation beam 104 through objective lens 130 and ontosample 120. The interaction of the excitation beams with a plurality offluorophores in the sample generates fluorescent emissions 105, 106,107, and 108. Fluorescent emissions 105, 106, 107, and 108 are reflectedby dichroic wedge 141 and transmitted through tube lens 115 and furtherthrough bandpass filter 145. Fluorescent emission 105 is transmittedtowards sensor array 151, fluorescent emission 106 is transmittedtowards sensor array 152, fluorescent emission 107 is transmittedtowards sensor array 153, and fluorescent emission 108 is transmittedtowards sensor array 154. In some implementations, mirrors 111 and 112can be oriented relative to the first excitation beam 103 and the secondexcitation beam 104, respectively, such that the first excitation beam103 and the second excitation beam 104 are spatially separated. This canallow the first excitation beam 103 to impinge onto a first location ofthe sample 120, and the second excitation beam 104 to impinge onto asecond location of the sample 220. As a result, fluorescent emissions105 and 106 (generated by the excitation beam 103) are spatiallyseparate from emissions 107 and 108 (generated by the excitation beam204). In embodiments, the first excitation beam 103 and the secondexcitation beam 104 are spatially separated by about 10 μm to about 500μm. In embodiments, the first excitation beam 103 and the secondexcitation beam 104 are spatially separated by about 10 μm, about 20 μm,about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about80 μm, about 90 μm, or about 100 μm. In embodiments, the firstexcitation beam 103 and the second excitation beam 104 are spatiallyseparated by about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm,about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. Inembodiments, the first excitation beam 103 and the second excitationbeam 104 are spatially separated by about 30 μm, about 35 μm, about 40μm, about 45 μm, about 50 μm, about 55 μm, or about 60 μm. Inembodiments, the first excitation beam 103 and the second excitationbeam 104 are spatially separated by about 50 μm. In embodiments, thefirst excitation beam 103 and the second excitation beam 104 arespatially separated by about 50 μm, about 51 μm, about 52 μm, about 53μm, about 54 μm, about 55 μm, about 56 μm, about 57 μm, about 58 μm,about 59 μm, or about 60 μm.

In a second configuration workflow, as shown in FIG. 2 , a first lightsource 201 provides a first excitation beam 203 and a second lightsource 202 provides a second excitation beam 204. Excitation beam 203 isdirected by mirror 212, mirror 213, and lens 210 towards dichroic filter240. Excitation beam 204 is directed by mirror 211, mirror 213, and lens210 towards dichroic filter 240. Dichroic filter 240 directs excitationbeam 203 and excitation beam 204 through objective lens 230 and ontosample 220. The interaction of the excitation beams with a plurality offluorophores in the sample generates fluorescent emissions 205, 206,207, and 208. Fluorescent emissions 205 and 207 are reflected bydichroic beamsplitter 241 through tube lens 215 and further throughbandpass filter 245. Fluorescent emission 205 is transmitted towardssensor array 251 and fluorescent emission 207 is transmitted towardssensor array 253. Fluorescent emissions 206 and 208 are reflected bydichroic beamsplitter 241 through tube lens 216 and further throughbandpass filter 246. Fluorescent emission 206 is transmitted towardssensor array 252 and fluorescent emission 208 is transmitted towardssensor array 254. In some implementations, mirrors 211 and 212 can beoriented relative to the first excitation beam 203 and the secondexcitation beam 204, respectively, such that the first excitation beam203 and the second excitation beam 204 are spatially separated. This canallow the first excitation beam 203 to impinge onto a first location ofthe sample 220, and the second excitation beam 204 to impinge onto asecond location of the sample 220. As a result, fluorescent emissions205 and 206 (generated by the excitation beam 203) are spatiallyseparate from emissions 207 and 208 (generated by the excitation beam204).

In embodiments, the objective lens is a microscope objective lens.Exemplary telecentric objective lenses useful in the invention includethose that are described in U.S. Pat. No. 5,847,400, which isincorporated herein by reference. In embodiments, the objective lens isan air objective lens. In embodiments, the objective lens is animmersion objective lens. In embodiments, the objective lens has a largenumerical aperture (NA) (e.g., NA in a range between 0.95 and 1.5) andperforms imaging via air immersion or liquid immersion (e.g., such aswater, oil, or other immersion fluids). For example, the NA may be atleast about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher. Thoseskilled in the art will appreciate that NA, being dependent upon theindex of refraction of the medium in which the lens is working, may behigher including, for example, up to 1.0 for air, 1.33 for pure water,or higher for other media such as oils. However, other embodiments mayhave lower NA values than the examples listed above. Image data obtainedby the optical assembly may have a resolution that is between 0.1 and 50microns or, more particularly, between 0.1 and 10 microns. Inembodiments, the numerical aperture for the camera is at least 0.2. Inembodiments, the numerical aperture for the camera is no greater than0.8. In embodiments, the numerical aperture for the camera is no greaterthan 0.5. Image systems described herein may have a resolution that issufficient to individually resolve the features or sites that areseparated by a distance of less than 10 μm, 5 μm, 2 μm, 1.5 μm, 1.0 μm,0.8 μm, 0.5 μm, or less. In embodiments, the image systems describedherein may have a resolution that is sufficient to individually resolvethe features or sites that are separated by a distance of 100 μm atmost.

In embodiments, each camera includes at least one CCD-CMOS sensor array.The light sensitive pixels are CCD, allowing for noiseless chargetransfer from line to line. The readout circuitry is CMOS, making itpossible to read out the columns in parallel and allowing high linerate. Each line successively captures a snapshot of the object as itpasses, allowing the accumulation of multiple images that can result ina very low noise image of a dark or hard-to-image object. When combinedwith the power efficiency of CMOS technology, a CCD-CMOS sensor permitsultra-high-speed imaging with excellent light sensitivity.

In embodiments, the CCD-CMOS sensor array is at least 8,000 pixels wide.In embodiments, the CCD-CMOS sensor array is at least 64 pixels long. Inembodiments, the CCD-CMOS sensor array is at least 128 pixels long. Inembodiments, each pixel has a width and a height in two dimensions(e.g., corresponding to an x and y axis). In embodiments, each pixel is3 μm wide. In embodiments, each pixel is 4 μm wide. In embodiments, eachpixel is 5 μm wide. In embodiments, each pixel is 2 μm wide. Inembodiments, each pixel is 1 μm wide. In embodiments, each pixel is 3 μmtall. In embodiments, each pixel is 4 μm tall. In embodiments, eachpixel is 5 μm tall. In embodiments, each pixel is 2 μm tall. Inembodiments, each pixel is 1 μm tall. In embodiments, the sensor arrayincludes a plurality of pixels. In embodiments, each pixel isapproximately square (i.e., each pixel has equal horizontal and verticalsampling pitch). In embodiments, each pixel is approximately rectangular(i.e., each pixel has unequal horizontal and vertical sampling pitch,resulting in an oblong shape).

In embodiments, the sensor array generates an output signal. The outputsignal of a image sensor is derived from the electrons generated bylight incident (e.g., emission) on the photodiode in each pixel. Theoutput voltage of the output signal depends on the transfer of thesignal electrons from the photodiode to the readout node. The shape andsize of the photodiode is known to control the rate of charge transfer.For example, in large photodiodes it is difficult to fully extract thegenerated electrons because of the reduction in lateral electric fieldthat pushes the electrons to the transfer transistor. Modulating theshape of the photodiode is known to enhance the transfer (e.g., atriangular shaped photodiode increases the lateral electric field).

In embodiments, the CCD-CMOS sensor array consists of rows of pixelsextending transverse to the scan direction, and has the capability totransfer charge from one row to the next at a rate determined by aninput synchronization signal. The synchronization signal is generated bythe position encoder of the motion stage that moves the object to beimaged (e.g., the sample) under the imaging lens. For example, thesample is translated under the objective at a constant speed. Theexcitation source illuminates a rectangular area on the sample. As thescan progresses, the fluorescence image of a particular object in thesample is translated on the camera chip (e.g., CCD-CMOS sensor array) atthe rate of magnification times the stage scan rate. In embodiments, thesample is scanned at about 1 mm²/sec, 1.5 mm²/sec, 5 mm²/sec, 10mm²/sec, 50 mm²/sec or 100 mm²/sec. In embodiments, the sample isscanned at 10 mm²/sec, 20 mm²/sec, 30 mm²/sec, 40 mm²/sec, or 50mm²/sec. In embodiments, the sample is scanned at least 20 mm²/sec.

In embodiments, the system may further include a scanning element, whichmay be a mechanical, electro-mechanical component, software component,or combination thereof configured to scan the sample along a direction,which may correspond to a scan direction. In an embodiment, the scandirection is orthogonal to the excitation direction of the sample. In anembodiment, the scan direction is non-orthogonal to the excitation beamdirection, wherein the orthogonal projected component directlycontributes to the final image reconstruction. The term “scanningelement” is intended to mean an element capable of sequentiallydetecting different portions of a sample. A scanning element canoperate, by changing the position of one or more components of thesystem including, for example, the light source the objective lens, theimage sensor, or the sample. Exemplary scanning elements include, butare not limited to a galvanometer configured to move a beam (e.g.,excitation beam) across a sample or a translation stage configured tomove the sample across the beam.

The TDI array (e.g., CCD-CMOS sensor array) imaging systems may also beconfigured to sequentially detect different portions of a sample bydifferent subsets of elements of the detector array, wherein transfer ofcharge between the subsets of elements proceeds at a rate synchronizedwith and in the same direction as the apparent motion of the samplebeing imaged. For example, CCD-CMOS sensor array imaging systems mayscan a sample such that a frame transfer device produces a continuousvideo image of the sample by means of a stack of linear arrays alignedwith and synchronized to the apparent movement of the sample, whereby asthe image moves from one line to the next, the stored charge moves alongwith it. Accumulation of charge can integrate during the entire timerequired for the row of charge to move from one end of the detector tothe serial register (or to the storage area of the device, in the caseof a frame transfer CCD).

The system may also include other components, including a collection oflenses (such as a collimating lens, a beam shaping lens (e.g., Powelllens), and a cylindrical lens), mirrors (e.g., a dichromatic mirror),beam splitter, one or more pinhole apertures, excitation filter, orcombinations thereof. For example, the direction, size, and/orpolarization of the light source may be adjusted by using lenses,mirrors, and/or polarizers. In embodiments, one or more of thecomponents of the system may be adjusted or manipulated automatically.Automatic control devices may include a motorized translation stage, anactuation device, one or more piezo stages, and/or one or more automaticswitch and flip mirrors and lenses. In embodiments, the system includesone or more optical components (e.g., a beam shaping lens) configured toshape the light emitted from the one or more light sources into desiredpatterns. For example, in some embodiments, the optical components mayshape the light into line patterns (e.g., by using one or more Powelllenses, or other beam shaping lenses, diffractive, or scatteringcomponents). In embodiments, the optical component includes a linegenerator.

In embodiments, the optical components include a Powell lens, amicro-lens, or micro-lens array. In embodiments, the optical componentincludes a micro-lens fabricated on glass, metal, or plastic. Inembodiments, the excitation beams may be directed through a beam shapinglens or lenses. In some embodiments, a single beam shaping lens may beused to shape the excitation beams output from a plurality light sources(e.g., 2 light sources). In some embodiments, a separate beam shapinglens may be used for each light beam. In embodiments, the beam shapinglens is a Powell lens, alternatively referred to as a Powell prism. Theshape of the beam may be shaped into an appropriate geometry accordingto known techniques, e.g., a line, conical, super-Gaussian, ring,doughnut, Bessel-Gauss, Hermite-Gaussian, Laguerre-Gaussian,Hypergeometric-Gaussian, Ince-Gaussian, and the like. In embodiments,the beam is uniform within acceptable limits (e.g., less than 30%intensity variation across the beam). In embodiments, the beam isprofiled or includes a gradient.

A sample refers to an object or location that is intended to bedetected. In embodiments, a sample includes a plurality of distinctfeatures that are targets for imaging. In some embodiments a sampleincludes a non-planar structure with a surface, such as a bead or awell, to which target nucleic acids have been attached as the targetfeatures. In embodiments, the sample is held by a sample holder. Thesample holder can be a multiwell plate. In some instances, the multiwellplate has 16, 24, 48, 96, 384 or more sample wells. In some of theseinstances, an array of light sources, e.g., LEDs, has 16, 24, 48, 96,384 or more corresponding light sources. In some instances, themultiwell plate is a standard microwell plate for biological analysis.

In an aspect is provided a nucleic acid sequencing system, wherein thegenetic sequencing system includes the imaging system as describedherein. Genetic sequencing systems utilize excitation beams to excitelabeled nucleotides in the DNA containing sample to enable analysis ofthe base pairs present within the DNA. High speed sequencing employshigh velocity scanning to deliver excitation beams to the DNAfluorophores, to stimulate sufficient emission of reactive photons fromthe DNA sample to be detected by the image sensors. Many of thenext-generation sequencing (NGS) technologies use a form of sequencingby synthesis (SBS), wherein modified nucleotides are used along with anenzyme to read the sequence of DNA templates in a controlled manner. Inembodiments, sequencing comprises a sequencing by synthesis process,where individual nucleotides are identified iteratively, as they arepolymerized to form a growing complementary strand. In embodiments,nucleotides added to a growing complementary strand include both a labeland a reversible chain terminator that prevents further extension, suchthat the nucleotide may be identified by the label before removing theterminator to add and identify a further nucleotide. Such reversiblechain terminators include removable 3′ blocking groups, for example asdescribed in U.S. Pat. Nos. 10,738,072, 7,541,444 and 7,057,026. Oncesuch a modified nucleotide has been incorporated into the growingpolynucleotide chain complementary to the region of the template beingsequenced, there is no free 3′-OH group available to direct furthersequence extension and therefore the polymerase cannot add furthernucleotides. Once the identity of the base incorporated into the growingchain has been determined, the 3′ reversible terminator may be removedto allow addition of the next successive nucleotide. In embodiments, thegenetic sequencing system utilizes the detection of four differentnucleotides that comprise four different labels.

In embodiments, the nucleic acid sequencing system utilizes thedetection of four different nucleotides using fewer than four differentlabels. As a first example, a pair of nucleotide types can be detectedat the same wavelength, but distinguished based on a difference insignal states, such as the intensity, for one member of the paircompared to the other, or based on a change to one member of the pair(e.g. via chemical modification, photochemical modification or physicalmodification) that causes apparent signal to appear or disappearcompared to the signal detected for the other member of the pair. As asecond example, three of four different nucleotide types can be detectedunder particular conditions while a fourth nucleotide type lacks a labelthat is detectable under those conditions, or is minimally detectedunder those conditions. Incorporation of the first three nucleotidetypes into a nucleic acid can be determined based on presence of theirrespective signals and incorporation of the fourth nucleotide type intothe nucleic acid can be determined based on absence or minimal detectionof any signal. As a third example, one nucleotide type can includelabel(s) that are detected in two different channels, whereas othernucleotide types are detected in no more than one of the channels.

In an aspect is provided a cell imaging system, wherein the cell imagingsystem includes the imaging system as described herein. Cell imagingsystems utilize excitation beams to detect emissions (e.g., diffractedlight, reflected light, refracted light) from a sample comprising a cell(e.g., a sample from a tissue of interest, or from a biopsy, bloodsample, or cell culture. Non-limiting examples of samples comprising acell include fluid or tissue from a subject, including, withoutlimitation, blood or a blood product (e.g., serum, plasma, platelets,buffy coats, or the like), umbilical cord blood, chorionic villi,amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g.,lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample,celocentesis sample, cells (blood cells, lymphocytes, placental cells,stem cells, bone marrow derived cells, embryo or fetal cells) or partsthereof (e.g., mitochondrial, nucleus, extracts, or the like), urine,feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen,lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the likeor combinations thereof. Non-limiting examples of tissues include organtissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder,reproductive organs, intestine, colon, spleen, brain, the like or partsthereof), epithelial tissue, hair, hair follicles, ducts, canals, bone,eye, nose, mouth, throat, ear, nails, the like, parts thereof orcombinations thereof. A sample may comprise cells or tissues that arenormal, healthy, diseased (e.g., infected), and/or cancerous (e.g.,cancer cells). A sample obtained from a subject may comprise cells orcellular material (e.g., nucleic acids) of multiple organisms (e.g.,virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasitenucleic acid).

In an aspect is provided a tissue imaging system, wherein the tissueimaging system includes the imaging system as described herein. Tissueimaging systems utilize excitation beams to detect emissions (e.g.,diffracted light, reflected light, refracted light) from a samplecomprising a tissue (e.g., a sample from a tissue of interest, or from abiopsy, blood sample, or cell culture).

In embodiments, the system (e.g., the nucleic acid sequencing system,the cell imaging system, or the tissue imaging system) includes anintegrated system of one or more interconnected chambers, ports, andchannels in fluid communication and configured for carrying out ananalytical reaction or process, either alone or in cooperation with anappliance or instrument that provides support functions. The reagentaspiration manifold and/or the reagent dispense manifold are in fluidiccommunication with a fluidic system. The fluid system may store fluidsfor washing or cleaning the fluidic network of the device, and also fordiluting the reactants. For example, the fluid system may includevarious reservoirs to store reagents, enzymes, other biomolecules,buffer solutions, aqueous, and non-polar solutions. Furthermore, thefluid system may also include waste reservoirs for receiving wasteproducts. As used herein, fluids may be liquids, gels, gases, or amixture of thereof. Also, a fluid can be a mixture of two or morefluids. The fluidic network may include a plurality of fluidiccomponents (e.g., fluid lines, pumps, aspirators, nozzles, valves, orother fluidic devices, manifolds, reservoirs) configured to have one ormore fluids flowing therethrough. In embodiments, the system includesone or more peristaltic pumps. In embodiments, the system includes oneor more syringe pumps. In embodiments, the support functions include atleast one of sample introduction, fluid and/or reagent driving means,temperature control, detection systems, data collection and integrationsystems, and are configured to determine the nucleic acid sequence of atemplate polynucleotide (e.g., a target polynucleotide, optionallycomprising a barcode). The device can use pressure drive flow control,e.g., utilizing valves and pumps, to manipulate the flow of reagents,molecules, or enzymes in one or more directions and/or into one or morechannels of a device.

In an aspect is provided a method of imaging a cell sample (e.g., atissue sample comprising a cell). In embodiments, the method includesproviding a sample comprising a cell, illuminating the sample using theimaging system described herein, and detecting emissions from the sample(e.g., fluorescent excitation events, scattered light, transmittedlight, or reflected light) at an active-pixel sensor array, and scanningthe sample in a synchronized manner (i.e., the transfer of both thefirst and the second charge is synchronized with the sample stagespeed).

In embodiments, the method further incudes a step of obtaining atwo-dimensional or three-dimensional picture, image, video, or otherrepresentation of the physical form or structure of the sample. Thisrepresentation can be obtained via light field, fluorescence, or othermicroscopic techniques. In embodiments, the method further includes anadditional imaging modality or immunohistochemistry modality (e.g.,immunostaining). Immunohistochemistry (IHC) is a powerful technique thatexploits the specific binding between an antibody and antigen to detectand localize specific antigens in cells and tissue, commonly detectedand examined with the light microscope. Known IHC modalities may beused, such as the protocols described in Magaki, S., Hojat, S. A., Wei,B., So, A., & Yong, W. H. (2019). Methods in molecular biology (Clifton,N.J.), 1897, 289-298, which is incorporated herein by reference. Inembodiments, the additional imaging modality includes bright fieldmicroscopy, phase contrast microscopy, Nomarskidifferential-interference-contrast microscopy, or dark field microscopy.In embodiments, the method further includes determining the cellmorphology (e.g., the cell boundary or cell shape) of a samplecomprising one or more cells. For example, determining the cell boundaryincludes comparing the pixel values of an image to a single intensitythreshold, which may be determined quickly using histogram-basedapproaches as described in Carpenter, A. et al Genome Biology 7, R100(2006) and Arce, S., Sci Rep 3, 2266 (2013)). Comparison of thisrepresentation with spatially resolved nucleic acid detection resultscan be used to localize genetic information with recognizable featuresof a tissue. Exemplary methods for spatial detection of nucleic acidsthat can be modified for use in the system and methods set forth hereinare described in US 2014/0066318 which is incorporated herein byreference. In embodiments, the method includes obtaining two-dimensionalplanes of images by scanning along one axis (e.g., the z direction). Forexample, multiple two-dimensional planes may be acquired for the samesample in the xy plane whereby detection events may be occurring ondifferent z-planes. In embodiments of the methods provided herein, themethod includes imaging through each of the multiple two-dimensionalplanes at a resolution sufficient to distinguish one imaged plane froman adjacent imaged plane. In embodiments, the methods and devicesdescribed herein simultaneously obtain a plurality of depth-resolvedoptically sectioned images.

In embodiments, the method includes performing an additional imageprocessing techniques (e.g., filtering, masking, smoothing, UnSharp Maskfilter (USM), deconvolution, or maximum intensity projection (MIP)). Inembodiments, the method includes computationally filtering the emissionsusing a linear or nonlinear filter that amplifies the high-frequencycomponents of the emission. For example, USM method applies a Gaussianblur to a duplicate of the original image and then compares it to theoriginal. If the difference is greater than a threshold setting, theimages are subtracted. In embodiments, the method includes a maximumintensity projection (MIP). A maximum intensity projection is avisualization technique that takes three-dimensional data (e.g.,emissions from varying depths within the sample) and turns it into asingle two-dimensional image. For example, the projection takes thebrightest pixel (voxel) in each depth and displays that pixel intensityvalue in the final two-dimensional image. Various machine learningapproaches may be used, for example, the methods described in Lugagne etal. Sci Rep 8, 11455 (2018) and Pattarone, G., et al. Sci Rep 11, 10304(2021), each of which is incorporated herein by reference. Inembodiments, the method includes focus stacking (e.g., z-stacking) whichcombines multiple images taken at different focus distances to give aresulting image with a greater depth of field (DOF) than any of theindividual source images.

One or more aspects or features of the subject matter described hereinmay be realized in digital electronic circuitry, integrated circuitry,specially designed ASICs (application specific integrated circuits),computer hardware, firmware, software, and/or combinations thereof.These various implementations may include implementation in one or morecomputer programs that are executable and/or interpretable on aprogrammable system including at least one programmable processor, whichmay be special or general purpose, coupled to receive data andinstructions from, and to transmit data and instructions to, a storagesystem, at least one input device (e.g., mouse, touch screen, etc.), andat least one output device. The methods and systems described herein canbe implemented or performed by a machine, such as a processor configuredwith specific instructions, a digital signal processor (DSP), anapplication specific integrated circuit (ASIC), a field programmablegate array (FPGA) or other programmable logic device, discrete gate ortransistor logic, discrete hardware components, or any combinationthereof designed to perform the functions described herein. A processorcan be a microprocessor. A processor can also be implemented as acombination of computing devices, e.g., a combination of a DSP and amicroprocessor, a plurality of microprocessors, one or moremicroprocessors in conjunction with a DSP core, or any other suchconfiguration. The elements of a method or process as described hereincan be implemented within computational hardware, in a software moduleexecuted by a processor, or in a combination of the two. A softwaremodule can reside in RAM memory, flash memory, ROM memory, EPROM memory,EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or anyother form of computer-readable storage medium known in the art.

The computer programs, which can also be referred to programs, software,software applications, applications, components, or code, includemachine instructions for a programmable processor, and can beimplemented in a high-level procedural language, an object-orientedprogramming language, a functional programming language, a logicalprogramming language, and/or in assembly/machine language. As usedherein, the term “machine-readable medium” refers to any computerprogram product, apparatus and/or device, such as for example magneticdiscs, optical disks, memory, and Programmable Logic Devices (PLDs),used to provide machine instructions and/or data to a programmableprocessor, including a machine-readable medium that receives machineinstructions as a machine-readable signal. The term “machine-readablesignal” refers to any signal used to provide machine instructions and/ordata to a programmable processor. The machine-readable medium can storesuch machine instructions non-transitorily, such as for example as woulda non-transient solid state memory or a magnetic hard drive or anyequivalent storage medium. The machine-readable medium can alternativelyor additionally store such machine instructions in a transient manner,such as for example as would a processor cache or other random accessmemory associated with one or more physical processor cores. Thecomputer can run any one of a variety of operating systems, such as forexample, any one of several versions of Windows, or of MacOS, or ofUnix, or of Linux.

With certain aspects, to provide for interaction with a user, thesubject matter described herein can be implemented on a computer havinga display device, such as for example a cathode ray tube (CRT) or aliquid crystal display (LCD) monitor for displaying information to theuser and a keyboard and a pointing device, such as for example a mouseor a trackball, by which the user may provide input to the computer.Other kinds of devices can be used to provide for interaction with auser as well. For example, feedback provided to the user can be any formof sensory feedback, such as for example visual feedback, auditoryfeedback, or tactile feedback; and input from the user may be receivedin any form, including, but not limited to, acoustic, speech, or tactileinput. Other possible input devices include, but are not limited to,touch screens or other touch-sensitive devices such as single ormulti-point resistive or capacitive trackpads, voice recognitionhardware and software, optical scanners, optical pointers, digital imagecapture devices and associated interpretation software, and the like.

The subject matter described herein may be implemented in a computingsystem that includes a back-end component (e.g., as a data server), orthat includes a middleware component (e.g., an application server), orthat includes a front-end component (e.g., a client computer having agraphical user interface or a Web browser through which a user mayinteract with an implementation of the subject matter described herein),or any combination of such back-end, middleware, or front-endcomponents. The components of the system may be interconnected by anyform or medium of digital data communication (e.g., a communicationnetwork). Examples of communication networks include a local areanetwork (“LAN”), a wide area network (“WAN”), the Internet, WiFi (IEEE802.11 standards), NFC, BLUETOOTH, ZIGBEE, and the like.

The computing system may include clients and servers. A client andserver are generally remote from each other and typically interactthrough a communication network. The relationship of client and serverarises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other.

The subject matter described herein can be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. The implementations set forth in the foregoingdescription do not represent all implementations consistent with thesubject matter described herein. Instead, they are merely some examplesconsistent with aspects related to the described subject matter.Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations can be provided in addition to those set forth herein.For example, the implementations described above can be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flow(s) depicted in theaccompanying figures and/or described herein do not necessarily requirethe particular order shown, or sequential order, to achieve desirableresults. Other implementations may be within the scope of the followingclaims.

EMBODIMENTS

Embodiment 1. An imaging system comprising: a sample stage moving at asample stage speed, wherein the sample stage is configured to receive asample comprising a first fluorophore and a second fluorophore; a firstsensor array and a second sensor array; a first light source configuredto provide a first excitation beam and a second light source configuredto provide a second excitation beam; a first optical system configuredto direct a first excitation beam and second excitation beam onto asample, wherein the interaction of the first excitation beam with thefirst fluorophore generates a first fluorescent emission, and theinteraction of the second excitation beam with the second fluorophoregenerates a second fluorescent emission; a second optical systemconfigured to direct the first fluorescent emission to the first sensorarray, and the second fluorescent emission to the second sensor array,wherein the first fluorescent emission impinges upon and generates afirst charge that transfers across the first sensor array, wherein thesecond fluorescent emission impinges upon and generates a second chargethat transfers across the second sensor array; and wherein the transferof both the first and the second charge is synchronized with the samplestage speed.

Embodiment 2. The imaging system of Embodiment 1, further comprising: athird fluorophore, and a fourth fluorophore; a third sensor array, and afourth sensor array, wherein the interaction of the first excitationbeam with the third fluorophore generates a third fluorescent emission,and the interaction of the second excitation beam with a fourthfluorophore generates a fourth fluorescent emission; wherein the secondoptical system is configured to direct the third fluorescent emission tothe third sensor array and the fourth fluorescent emission to the fourthsensor array, wherein the third fluorescent emission impinges upon andgenerates a third charge that transfers across the third sensor array,wherein the fourth fluorescent emission impinges upon and generates afourth charge that transfers across the fourth sensor array, wherein thetransfer of at least one of the third and the fourth charge issynchronized with the sample stage speed.

Embodiment 3. An imaging system comprising: a sample stage moving at asample stage speed, wherein the sample stage comprises a samplecomprising a first fluorophore, a second fluorophore, a thirdfluorophore, and a fourth fluorophore; a first sensor array, secondsensor array, third sensor array, and a fourth sensor array; a firstlight source configured to provide a first excitation beam and a secondlight source configured to provide a second excitation beam; a firstoptical system configured to direct a first excitation beam and secondexcitation beam onto a sample, wherein the interaction of the firstexcitation beam with the first fluorophore generates a first fluorescentemission, the interaction of the second excitation beam with the secondfluorophore generates a second fluorescent emission, the interaction ofthe first excitation beam with a third fluorophore generates a thirdfluorescent emission, and the interaction of the second excitation beamwith a fourth fluorophore generates a fourth fluorescent emission; asecond optical system configured to direct the first fluorescentemission to the first sensor array, the second fluorescent emission tothe second sensor array, the third fluorescent emission to the thirdsensor array, the fourth fluorescent emission to the fourth sensorarray, wherein the first fluorescent emission impinges upon andgenerates a first charge that transfers across the first sensor array,wherein the second fluorescent emission impinges upon and generates asecond charge that transfers across the second sensor array, wherein thethird fluorescent emission impinges upon and generates a third chargethat transfers across the third sensor array, wherein the fourthfluorescent emission impinges upon and generates a fourth charge thattransfers across the fourth sensor array, wherein the transfer of atleast one of the first, the second, the third and the fourth charge issynchronized with the sample stage speed.

Embodiment 4. The imaging system of any one Embodiments 1 to 3, whereinthe first optical system is configured to direct the first excitationbeam to a first region of the sample and direct the second excitationbeam to a second region of the sample, wherein the first region andsecond region are separated by at least about 10 μm to about 500 μm.

Embodiment 5. The imaging system of Embodiments 2 to 4, wherein thesecond optical system comprises a first optical element including: afirst surface configured to reflect the first fluorescent emissiontowards the first sensor array, and reflect the third fluorescentemission towards the third sensor array; and a second surface configuredto reflect the second fluorescent emission towards the second sensorarray, and reflect the fourth fluorescent emission towards the fourthsensor array.

Embodiment 6. The imaging system of Embodiment 5, wherein the secondoptical system comprises a second optical element downstream from thefirst optical element and configured to focus the first fluorescentemission, the second fluorescent emission, the third fluorescentemission, and the fourth fluorescent emission.

Embodiment 7. The imaging system of Embodiment 6, wherein the secondoptical system comprises a band pass filter configured to selectivelytransmit the first fluorescent emission, the second fluorescentemission, the third fluorescent emission, and the fourth fluorescentemission.

Embodiment 8. The imaging system of Embodiment 7, wherein a detectioncamera includes the first sensor array, the second sensor array, thethird sensor array, and the fourth sensor array.

Embodiment 9. The imaging system of Embodiment 5, wherein the firstoptical element is a dichroic wedge.

Embodiment 10. The imaging system of Embodiments 2 to 4, wherein thesecond optical system comprises a first optical element configured toreflect the first fluorescent emission towards the first sensor array,and reflect the third fluorescent emission towards the third sensorarray; and transmit the second fluorescent emission towards the secondsensor array, and transmit the fourth fluorescent emission towards thefourth sensor array.

Embodiment 11. The imaging system of Embodiment 10, wherein the secondoptical system comprises: a first lens downstream from the first opticalelement and configured to focus the first fluorescent emission and thethird fluorescent emission; and a second lens downstream from the firstoptical element and configured to focus the second fluorescent emissionand the fourth fluorescent emission.

Embodiment 12. The imaging system of Embodiment 11, wherein the secondoptical system comprises: a first band pass filter configured toselectively transmit the first fluorescent emission and the thirdfluorescent emission; and a second band pass filter configured toselectively transmit the second fluorescent emission and the fourthfluorescent emission.

Embodiment 13. The imaging system of Embodiment 12, wherein a firstdetection camera includes the first sensor array and the third sensorarray, and a second detection camera includes the second sensor arrayand the fourth sensor array.

Embodiment 14. The imaging system of Embodiment 10, wherein the firstoptical element is a dichroic beamsplitter.

Embodiment 15. The imaging system of one of Embodiments 1 to 14, whereineach sensor array is a TDI sensor array.

Embodiment 16. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 1,000 to 20,000 pixels wide.

Embodiment 17. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 3,000 to 10,000 pixels wide.

Embodiment 18. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 5,000 to 8,000 pixels wide.

Embodiment 19. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 5,500 pixels wide.

Embodiment 20. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 1,000 to 20,000 pixels wide and about 10 to300 pixels long.

Embodiment 21. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 3,000 to 10,000 pixels wide and about 10 to300 pixels long.

Embodiment 22. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 5,000 to 10,000 pixels wide and about 30 to300 pixels long.

Embodiment 23. The imaging system of one of Embodiments 1 to 15, whereineach sensor array is about 8,000 pixels wide and about 128 pixels long.

Embodiment 24. The imaging system of one of Embodiments 1 to 23, whereinthe sample stage is a motorized translation stage.

Embodiment 25. The imaging system of one of Embodiments 1 to 24, whereinthe sample stage comprises a position encoder, wherein the positionencoder generates a synchronization signal that synchronizes thetransfer of the charges.

Embodiment 26. The imaging system of one of Embodiments 1 to 25, furthercomprising a collimating lens, a beam shaping lens, or a cylindricallens.

Embodiment 27. The imaging system of one of Embodiments 1 to 26, furthercomprising one or more line generators.

Embodiment 28. The imaging system of one of Embodiments 1 to 26, furthercomprising two line generators.

Embodiment 29. A method of imaging a sample comprising: a) directing afirst excitation beam and a second excitation beam onto a sample,wherein said sample is on a sample stage moving at a sample stage speed,wherein the sample comprises a first fluorophore that generates a firstfluorescent emission and a second fluorophore that generates a secondfluorescent emission following interaction with a first excitation beamand a second excitation beam, respectively; b) directing said firstfluorescent emission to impinge upon and generate a first charge thattransfers across a first sensor array at a first charge speed, anddirecting said second fluorescent emission to impinge upon and generatea second charge that transfers across a second sensor array at a secondcharge speed, wherein at least one of the first charge speed and thesecond charge speed is synchronized with the sample stage speed; and c)scanning the sample in a scan dimension and repeating step a) and stepb) to form an image of the sample.

Embodiment 30. The method of Embodiment 29, wherein the sample furthercomprises a third fluorophore that generates a third fluorescentemission and a fourth fluorophore that generates a fourth fluorescentemission following interaction with a first excitation beam and a secondexcitation beam, respectively; and directing said third fluorescentemission to impinge upon and generate a third charge that transfersacross a third sensor array at a third charge speed, and directing saidfourth fluorescent emission to impinge upon and generate a fourth chargethat transfers across a fourth sensor array at a fourth charge speed.

Embodiment 31. The method of Embodiment 30, wherein the method comprisesimaging the sample comprising four different fluorophoressimultaneously.

Embodiment 32. The method of Embodiments 30 or 31, wherein the firstexcitation beam and the second excitation beam comprise an excitationline.

Embodiment 33. The method of one of Embodiments 30 to 32, whereinscanning the sample comprises moving the sample stage.

Embodiment 34. The method of one of Embodiments 30 to 33, whereinscanning the sample comprises moving the sample stage at a constantspeed.

Embodiment 35. The method of one of Embodiments 30 to 34, furthercomprising storing a data representation of said image of said sample ina computer readable memory.

Embodiment 36. The method of one of Embodiments 30 to 35, wherein theexcitation beam comprises UV radiation, VIS radiation, or IR radiation.

Embodiment 37. The method of one of Embodiments 30 to 36, wherein theexcitation beam comprises excitation beams having a wavelength of 405nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

Embodiment 38. The method of one of Embodiments 30 to 37, wherein thesample comprises modified nucleotides.

Embodiment 39. The method of one of Embodiments 30 to 38, wherein thesample comprises an array having a plurality of individual sites.

I. Definitions

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

As used herein, the term “nucleic acid” refers to nucleotides (e.g.,deoxyribonucleotides or ribonucleotides) and polymers thereof in eithersingle-, double- or multiple-stranded form, or complements thereof. Theterms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, inthe usual and customary sense, to a sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, e.g., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA withlinear or circular framework. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

As used herein, the term “polynucleotide template” refers to anypolynucleotide molecule that may be bound by a polymerase and utilizedas a template for nucleic acid synthesis. As used herein, the term“polynucleotide primer” or “primer” refers to any polynucleotidemolecule that may hybridize to a polynucleotide template, be bound by apolymerase, and be extended in a template-directed process for nucleicacid synthesis, such as in a PCR or sequencing reaction. Polynucleotideprimers attached to a core polymer within a core are referred to as“core polynucleotide primers.”

In general, the term “target polynucleotide” refers to a nucleic acidmolecule or polynucleotide in a starting population of nucleic acidmolecules having a target sequence whose presence, amount, and/ornucleotide sequence, or changes in one or more of these, are desired tobe determined. In general, the term “target sequence” refers to anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA, miRNA, rRNA, or others. The target sequencemay be a target sequence from a sample or a secondary target such as aproduct of an amplification reaction. A target polynucleotide is notnecessarily any single molecule or sequence. For example, a targetpolynucleotide may be any one of a plurality of target polynucleotidesin a reaction, or all polynucleotides in a given reaction, depending onthe reaction conditions. For example, in a nucleic acid amplificationreaction with random primers, all polynucleotides in a reaction may beamplified. As a further example, a collection of targets may besimultaneously assayed using polynucleotide primers directed to aplurality of targets in a single reaction. As yet another example, allor a subset of polynucleotides in a sample may be modified by theaddition of a primer-binding sequence (such as by the ligation ofadapters containing the primer binding sequence), rendering eachmodified polynucleotide a target polynucleotide in a reaction with thecorresponding primer polynucleotide(s).

As used herein, the term “flow cell” refers to the reaction vessel in anucleic acid sequencing device. The flow cell is typically a glass slidecontaining small fluidic channels (e.g., a glass slide 75 mm×25 mm×1 mmhaving one or more channels), through which sequencing solutions (e.g.,polymerases, nucleotides, and buffers) may traverse. Though typicallyglass, suitable flow cell materials may include polymeric materials,plastics, silicon, quartz (fused silica), Borofloat® glass, silica,silica-based materials, carbon, metals, an optical fiber or opticalfiber bundles, sapphire, or plastic materials such as COCs and epoxies.The particular material can be selected based on properties desired fora particular use. For example, materials that are transparent to adesired wavelength of radiation are useful for analytical techniquesthat will utilize radiation of the desired wavelength. Conversely, itmay be desirable to select a material that does not pass radiation of acertain wavelength (e.g., being opaque, absorptive, or reflective). Inembodiments, the material of the flow cell is selected due to theability to conduct thermal energy. In embodiments, a flow cell includesinlet and outlet ports and a flow channel extending therebetween.

A “line generator” as used herein refers to an optical component that isconfigured to generate a diffraction-limited or near diffraction-limitedexcitation beam in the plane perpendicular to the optical axis ofpropagation with a substantially uniform intensity distribution alongthe horizontal axis of the line. Exemplary line generators include, butare not limited to, a one dimensional diffuser having angularuniformity, cylindrical micro-lens array, diffractive element oraspheric refractive lens such as a Powell lens.

As used herein, the term “substrate” refers to a solid support material.The substrate can be non-porous or porous. The substrate can be rigid orflexible. A nonporous substrate generally provides a seal against bulkflow of liquids or gases. Exemplary solid supports include, but are notlimited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics,resins, Zeonor, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses, optical fiberbundles, photopatternable dry film resists, UV-cured adhesives andpolymers. Particularly useful solid supports for some embodiments haveat least one surface located within a flow cell. The term “surface” isintended to mean an external part or external layer of a substrate. Thesurface can be in contact with another material such as a gas, liquid,gel, polymer, organic polymer, second surface of a similar or differentmaterial, metal, or coat. The surface, or regions thereof, can besubstantially flat. The substrate and/or the surface can have surfacefeatures such as wells, pits, channels, ridges, raised regions, pegs,posts or the like. The term “well” refers to a discrete concave featurein a substrate having a surface opening that is completely surrounded byinterstitial region(s) of the surface. Wells can have any of a varietyof shapes at their opening in a surface including but not limited toround, elliptical, square, polygonal, or star shaped (e.g., star shapedwith any number of vertices). The cross section of a well takenorthogonally with the surface may be curved, square, polygonal,hyperbolic, conical, or angular.

As used herein, the terms “sequencing”, “sequence determination”,“determining a nucleotide sequence”, and the like include determinationof a partial or complete sequence information (e.g., a sequence) of apolynucleotide being sequenced, and particularly physical processes forgenerating such sequence information. That is, the term includessequence comparisons, consensus sequence determination, contig assembly,fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofnucleotides in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. In some embodiments, a sequencing process describedherein comprises contacting a template and an annealed primer with asuitable polymerase under conditions suitable for polymerase extensionand/or sequencing. The sequencing methods are preferably carried outwith the target polynucleotide arrayed on a solid substrate within aflow cell (e.g., within a channel of the flow cell). In an embodiment,the sequencing is sequencing by synthesis (SBS). Briefly, SBS methodsinvolve contacting target nucleic acids with one or more labelednucleotides (e.g., fluorescently labeled) in the presence of a DNApolymerase. Optionally, the labeled nucleotides can further include areversible termination property that terminates extension once thenucleotide has been incorporated. Thus, for embodiments that usereversible termination, a cleaving solution can be delivered to the flowcell (before or after detection occurs). Washes can be carried outbetween the various delivery steps. The cycle can then be repeated ntimes to extend the primer by n nucleotides, thereby detecting asequence of length n. Exemplary SBS procedures and detection platformsthat can be readily adapted for use with the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008), WO 2004/018497; and WO 2007/123744, each of which isincorporated herein by reference in its entirety. In an embodiment,sequencing is pH-based DNA sequencing. The concept of pH-based DNAsequencing, has been described in the literature, including thefollowing references that are incorporated by reference: US2009/0026082;and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006) whichare incorporated herein by reference in their entirety. Other sequencingprocedures that use cyclic reactions can be used, such aspyrosequencing. Sequencing-by-ligation reactions are also usefulincluding, for example, those described in Shendure et al. Science309:1728-1732 (2005).

As used herein, the term “feature” refers a point or area in a patternthat can be distinguished from other points or areas according to itsrelative location. An individual feature can include one or morepolynucleotides. For example, a feature can include a single targetnucleic acid molecule having a particular sequence or a feature caninclude several nucleic acid molecules having the same sequence (and/orcomplementary sequence, thereof). Different molecules that are atdifferent features of a pattern can be differentiated from each otheraccording to the locations of the features in the pattern. Non-limitingexamples of features include wells in a substrate, particles (e.g.,beads) in or on a substrate, polymers in or on a substrate, projectionsfrom a substrate, ridges on a substrate, or channels in a substrate.

The term “image” is used according to its ordinary meaning and refers toa representation of all or part of an object. The representation may bean optically detected reproduction. For example, an image can beobtained from fluorescent, luminescent, scatter, or absorption signals.The part of the object that is present in an image can be the surface orother xy plane of the object. Typically, an image is a 2-dimensionalrepresentation of a 3 dimensional object. An image may include signalsat differing intensities (i.e., signal levels). An image can be providedin a computer readable format or medium. An image is derived from thecollection of focus points of light rays coming from an object (e.g.,the sample), which may be detected by any image sensor.

As used herein, the term “signal” is intended to include, for example,fluorescent, luminescent, scatter, or absorption impulse orelectromagnetic wave transmitted or received. Signals can be detected inthe ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range(about 391 to 770 nm), infrared (IR) range (about 0.771 to 25 microns),or other range of the electromagnetic spectrum. The term “signal level”refers to an amount or quantity of detected energy or coded information.For example, a signal may be quantified by its intensity, wavelength,energy, frequency, power, luminance, or a combination thereof. Othersignals can be quantified according to characteristics such as voltage,current, electric field strength, magnetic field strength, frequency,power, temperature, etc. Absence of signal is understood to be a signallevel of zero or a signal level that is not meaningfully distinguishedfrom noise.

The term “xy coordinates” refers to information that specifies location,size, shape, and/or orientation in an xy plane. The information can be,for example, numerical coordinates in a Cartesian system. Thecoordinates can be provided relative to one or both of the x and y axesor can be provided relative to another location in the xy plane (e.g., afiducial). The term “xy plane” refers to a 2 dimensional area defined bystraight line axes x and y. When used in reference to a detectingapparatus and an object observed by the detector, the xy plane may bespecified as being orthogonal to the direction of observation betweenthe detector and object being detected. The terms “z-axis” and “zdirection” are intended to be used consistently with their use in theart of microscopy and imaging systems in general, in which the z-axisrefers to the focal axis. Accordingly, a z-axis translation results inincreasing or decreasing the length of the focal axis. A z-axistranslation can be carried out, for example, by moving a sample stagerelative to an optical stage (e.g., by moving the sample stage or anoptical element or both).

As used herein, the terms “cluster” and “colony” are usedinterchangeably to refer to a discrete site on a solid support thatincludes a plurality of immobilized polynucleotides and a plurality ofimmobilized complementary polynucleotides. The term “clustered array”refers to an array formed from such clusters or colonies. In thiscontext the term “array” is not to be understood as requiring an orderedarrangement of clusters. The term “array” is used in accordance with itsordinary meaning in the art, and refers to a population of differentmolecules that are attached to one or more solid-phase substrates suchthat the different molecules can be differentiated from each otheraccording to their relative location. A flow cell may include an arrayand can include different molecules that are each located at differentaddressable features on a solid-phase substrate. The molecules of thearray can be nucleic acid primers, nucleic acid probes, nucleic acidtemplates or nucleic acid enzymes such as polymerases or ligases. Arraysuseful in the invention can have densities that ranges from about 2different features to many millions, billions or higher. The density ofan array can be from 2 to as many as a billion or more differentfeatures per square cm. For example an array can have at least about 100features/cm², at least about 1,000 features/cm², at least about 10,000features/cm², at least about 100,000 features/cm², at least about10,000,000 features/cm², at least about 100,000,000 features/cm², atleast about 1,000,000,000 features/cm², at least about 2,000,000,000features/cm² or higher. In embodiments, the arrays have features at anyof a variety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher.Clustering refers to the process of generating clusters (i.e.,solid-phase amplification of polynucleotides).

The term “nucleic acid sequencing device” means an integrated system ofone or more chambers, ports, and channels that are interconnected and influid communication and designed for carrying out an analytical reactionor process, either alone or in cooperation with an appliance orinstrument that provides support functions, such as sample introduction,fluid and/or reagent driving means, temperature control, detectionsystems, data collection and/or integration systems, for the purpose ofdetermining the nucleic acid sequence of a template polynucleotide.Nucleic acid sequencing devices may further include valves, pumps, andspecialized functional coatings on interior walls. Nucleic acidsequencing devices may include a receiving unit, or platen, that orientsthe flow cell such that a maximal surface area of the flow cell isavailable to be exposed to an optical lens. Other nucleic acidsequencing devices include those provided by Illumina™, Inc. (e.g.HiSeg™, MiSeg™, NextSeg™, or NovaSeg™ systems), Life Technologies™ (e.g.ABI PRISM™, or SOLiD™ systems), Pacific Biosciences (e.g. systems usingSMRT™ Technology such as the Sequel™ or RS II™ systems), or Qiagen (e.g.Genereader™ system). Nucleic acid sequencing devices may further includefluidic reservoirs (e.g., bottles), valves, pressure sources, pumps,sensors, control systems, valves, pumps, and specialized functionalcoatings on interior walls. In embodiments, the device includes aplurality of a sequencing reagent reservoirs and a plurality ofclustering reagent reservoirs. In embodiments, the clustering reagentreservoir includes amplification reagents (e.g., an aqueous buffercontaining enzymes, salts, and nucleotides, denaturants, crowdingagents, etc.) In embodiments, the reservoirs include sequencing reagents(such as an aqueous buffer containing enzymes, salts, and nucleotides);a wash solution (an aqueous buffer); a cleave solution (an aqueousbuffer containing a cleaving agent, such as a reducing agent); or acleaning solution (a dilute bleach solution, dilute NaOH solution,dilute HCl solution, dilute antibacterial solution, or water). The fluidof each of the reservoirs can vary. The fluid can be, for example, anaqueous solution which may contain buffers (e.g., saline-sodium citrate(SSC), ascorbic acid, tris(hydroxymethyl)aminomethane or “Tris”),aqueous salts (e.g., KCl or (NH4)2SO4)), nucleotides, polymerases,cleaving agent (e.g., tri-n-butyl-phosphine, triphenyl phosphine and itssulfonated versions (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), andtri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agentscavenger compounds (e.g., 2′-Dithiobisethanamine or11-Azido-3,6,9-trioxaundecane-1-amine), chelating agents (e.g., EDTA),detergents, surfactants, crowding agents, or stabilizers (e.g., PEG,Tween, BSA). Non-limited examples of reservoirs include cartridges,pouches, vials, containers, and eppendorf tubes. In embodiments, thedevice is configured to perform fluorescent imaging. In embodiments, thedevice includes one or more light sources (e.g., one or more lasers). Inembodiments, the illuminator or light source is a radiation source(i.e., an origin or generator of propagated electromagnetic energy)providing incident light to the sample. A radiation source can includean illumination source producing electromagnetic radiation in theultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about390 to 770 nm), or infrared (IR) range (about 0.77 to 25 microns), orother range of the electromagnetic spectrum. In embodiments, theilluminator or light source is a lamp such as an arc lamp or quartzhalogen lamp. In embodiments, the illuminator or light source is acoherent light source. In embodiments, the light source is a laser, LED(light emitting diode), a mercury or tungsten lamp, or asuper-continuous diode. In embodiments, the light source providesexcitation beams having a wavelength between 200 nm to 1500 nm. Inembodiments, the laser provides excitation beams having a wavelength of405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm,640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm. In embodiments, theilluminator or light source is a light-emitting diode (LED). The LED canbe, for example, an Organic Light Emitting Diode (OLED), a Thin FilmElectroluminescent Device (TFELD), or a Quantum dot based inorganicorganic LED. The LED can include a phosphorescent OLED (PHOLED). Inembodiments, the nucleic acid sequencing device includes an imagingsystem (e.g., an imaging system as described herein). The imaging systemcapable of exciting one or more of the identifiable labels (e.g., afluorescent label) linked to a nucleotide and thereafter obtain imagedata for the identifiable labels. The image data (e.g., detection data)may be analyzed by another component within the device. The imagingsystem may include a system described herein and may include afluorescence spectrophotometer including an objective lens and/or asolid-state imaging device. The solid-state imaging device may include acharge coupled device (CCD) and/or a complementary metal oxidesemiconductor (CMOS).

As used herein, the term “label” or “labels” generally refer tomolecules that can directly or indirectly produce or result in adetectable signal either by themselves or upon interaction with anothermolecule. A label moiety can be any moiety that allows the sample to bedetected, for example, using a spectroscopic method. Exemplary labelmoieties are fluorescent labels, mass labels, chemiluminescent labels,electrochemical labels, detectable labels and the like. Non-limitingexamples of detectable labels include labels comprising fluorescentdyes, biotin, digoxin, haptens, and epitopes. In general, a dye is amolecule, compound, or substance that can provide an opticallydetectable signal, such as a colorimetric, luminescent, bioluminescent,chemiluminescent, phosphorescent, or fluorescent signal. In embodiments,the dye is a fluorescent dye. Non-limiting examples of dyes, some ofwhich are commercially available, include CF dyes (Biotium, Inc.), AlexaFluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GEHealthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes(Anaspec, Inc.). In embodiments, the label is a fluorophore. Examples ofdetectable agents (e.g., labels) include imaging agents, includingfluorescent and luminescent substances, molecules, or compositions,including, but not limited to, a variety of organic or inorganic smallmolecules commonly referred to as “dyes,” “labels,” or “indicators.”Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, andcyanine dyes. In embodiments, the detectable moiety is a fluorescentmolecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye,phenanthridine dye, or rhodamine dye). In embodiments, the detectablemoiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye,fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye).

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The term “optical filter” refers to a device for selectively passing orrejecting passage of radiation in a wavelength, polarization, orfrequency dependent manner. In embodiments, an optical filter is adichroic filter or dielectric filter. The fluorescence from differentfluorophores could be further separated by dichromatic optical elementsand projected into spatially separated lines on the sensor array. Tofurther suppress the background from out-of-the-focus fluorescencesignal, an optical filter with multiple stripe patterns may be placed infront of the camera to pass only the selected fluorescence lines andreject the unwanted ones. An optical filter is used in accordance withits plain ordinary meaning in the art and refers to a device forselectively passing or rejecting the passage of light having aparticular wavelength, polarization or frequency. The term can includean interference filter in which multiple layers of dielectric materialspass or reflect light according to constructive or destructiveinterference between reflections from the various layers. Interferencefilters are also referred to in the art as dichroic filters, ordielectric filters. The term can include an absorptive filter whichprevents the passage of light having a selective wavelength orwavelength range by absorption. Absorptive filters include, for example,colored glass or liquid. A filter can have one or more particular filtertransmission characteristics including, for example, bandpass, shortpass and long pass. A band pass filter selectively passes light in awavelength range defined by a center wavelength of maximum radiationtransmission (T_(max)) and a bandwidth and blocks passage of lightoutside of this range. T_(max) defines the percentage of radiationtransmitted at the center wavelength. The bandwidth is typicallydescribed as the full width at half maximum (FWHM) which is the range ofwavelengths passed by the filter at a transmission value that is half ofT_(max). A band pass filter can have a FWHM of 10 nanometers (nm), 20nm, 30 nm, 40 nm or 50 nm. A long pass filter selectively passes higherwavelength light as defined by a T_(max) and a cut on wavelength. Thecut on wavelength is the wavelength at which light transmission is halfof T_(max), when the wavelength increases above the cut on wavelength,transmission percentage increases and as wavelength decreases below thecut on wavelength transmission percentage decreases. A short pass filterselectively passes lower wavelength radiation as defined by a T_(max)and a cut off wavelength. The cut off wavelength is the wavelength atwhich light transmission is half of T_(max); as wavelength increasesabove the cut off wavelength, transmission percentage decreases and aswavelength decreases below the cut off wavelength transmissionpercentage increases. A filter can have a T_(max) of 50-100%, 60-90% or70-80%.

The term “synchronize” is used in accordance with its ordinary meaningand refers to events that co-occur. For example, the sample stage isconfigured to synchronize with the charge transfer across the sensorarray, such that the sample stage is configured to move in concert withthe charge transfer. In embodiments, synchrony does not require thesample stage move at the same rate as the charge transfer. Inembodiments, the sample stage moves at a stage rate and the chargetransfers across the sensor array at a charge transfer rate, wherein thestage rate and the charge transfer rate are associated (e.g., the stagerate is a fraction or ratio of the charge transfer rate). Inembodiments, synchronized events contemporaneously occur. Inembodiments, the sample stage is configured to move at approximately thesame speed as the charge transfer. In embodiments, the charge transferrate is 1000 ns (nanoseconds)/μm, 500 ns (nanoseconds)/μm, 100 ns(nanoseconds)/μm, 50 ns (nanoseconds)/μm, or less (e.g., 40 ns(nanoseconds)/μm). In embodiments, the sample is scanned at about 1mm²/sec, 1.5 mm²/sec, 5 mm²/sec, 10 mm²/sec, 50 mm²/sec or 100 mm²/secwherein the scan rate is associated with the charge transfer rate.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. An imaging system comprising: a sample stagemoving at a sample stage speed, wherein the sample stage comprises asample; a first sensor array, and a second sensor array; a first opticalsystem configured to direct a first excitation beam and secondexcitation beam onto a sample; a second optical system configured todirect: a light emission from said sample to the first sensor array,thereby generating a first charge that transfers across the first sensorarray, a second light emission from said sample to the second sensorarray, thereby generating a second charge that transfers across thesecond sensor array, wherein the transfer of the first charge and thesecond charge is synchronized with the sample stage speed.
 2. Theimaging system claim 1, wherein the first optical system is configuredto direct the first excitation beam to a first region of the sample anddirect the second excitation beam to a second region of the sample,wherein the first region and second region are separated by about 10 μmto about 500 μm.
 3. The imaging system of claim 2, wherein the firstregion and second region are separated by about 30 μm to about 100 μm.4. The imaging system claim 1, wherein the second optical systemcomprises a first optical element including: a first surface configuredto reflect the first light emission towards the first sensor array; anda second surface configured to reflect the second light emission towardsthe second sensor array.
 5. The imaging system of claim 4, wherein thesecond optical system comprises a second optical element downstream fromthe first optical element and configured to focus the first lightemission and the second light emission.
 6. The imaging system of claim5, wherein the second optical system comprises a band pass filterconfigured to selectively transmit the first light emission and thesecond light emission.
 7. The imaging system of claim 1, wherein adetection camera includes the first sensor array and the second sensorarray.
 8. The imaging system claim 1, wherein each sensor array is a TDIsensor array.
 9. The imaging system claim 1, wherein each sensor arrayis about 1,000 to 20,000 pixels wide.
 10. The imaging system claim 1,wherein each sensor array is about 1,000 to 20,000 pixels wide and about10 to 300 pixels long.
 11. The imaging system claim 1, wherein eachsensor array is about 8,000 pixels wide and about 128 pixels long. 12.The imaging system claim 1, wherein the sample stage is a motorizedtranslation stage.
 13. The imaging system claim 1, wherein the samplestage comprises a position encoder, wherein the position encodergenerates a synchronization signal that synchronizes the transfer of thecharges.
 14. The imaging system claim 1, further comprising acollimating lens, a beam shaping lens, or a cylindrical lens.
 15. Theimaging system claim 1, further comprising one or more line generators.16. The imaging system of claim 1, further comprising a third sensorarray, and wherein the second optical system is configured to direct athird light emission from said sample to the third sensor array, therebygenerating a third charge that transfers across the third sensor array.17. The imaging system of claim 1, wherein the first light emission andthe second light emission comprise diffracted light, refracted light,scattered light, transmitted light, or reflected light.
 18. The imagingsystem of claim 1, wherein the first light emission and second lightemission is a fluorescent emission.
 19. The imaging system of claim 16,further comprising a fourth sensor array, and wherein the second opticalsystem is configured to direct: a fourth light emission from said sampleto the fourth sensor array, thereby generating a fourth charge thattransfers across the fourth sensor array, wherein the transfer of thefirst, second, third and fourth charge is synchronized with the samplestage speed.
 20. The imaging system of claim 19, wherein a firstdetection camera includes the first sensor array and the third sensorarray, and a second detection camera includes the second sensor arrayand the fourth sensor array.
 21. The imaging system of claim 19, whereinthe first optical element is a dichroic beamsplitter.
 22. The imagingsystem claim 19, wherein the second optical system comprises a firstoptical element configured to reflect the first light emission towardsthe first sensor array and reflect the third light emission towards thethird sensor array; and transmit the second light emission towards thesecond sensor array and transmit the fourth light emission towards thefourth sensor array.
 23. The imaging system of claim 22, wherein thesecond optical system comprises: a first lens downstream from the firstoptical element and configured to focus the first light emission and thethird light emission; and a second lens downstream from the firstoptical element and configured to focus the second light emission andthe fourth light emission.
 24. The imaging system of claim 22, whereinthe second optical system comprises: a first band pass filter configuredto selectively transmit the first light emission and the third lightemission; and a second band pass filter configured to selectivelytransmit the second light emission and the fourth light emission.
 25. Amethod of imaging a sample comprising: a) directing a first excitationbeam and a second excitation beam onto a sample, wherein said sample ison a sample stage moving at a sample stage speed, wherein the samplecomprises cell or biological tissue; b) directing a first light emissionfrom said sample to impinge upon and generate a first charge thattransfers across a first sensor array at a first charge speed, anddirecting a second light emission from said sample to impinge upon andgenerate a second charge that transfers across a second sensor array ata second charge speed, wherein at least one of the first charge speedand the second charge speed is synchronized with the sample stage speed;and c) scanning the sample in a scan dimension and repeating step a) andstep b) to form an image of the sample.
 26. The method of claim 25,wherein the sample further comprises a third light emission and a fourthlight emission; and directing said third light emission to impinge uponand generate a third charge that transfers across a third sensor arrayat a third charge speed, and directing said fourth light emission toimpinge upon and generate a fourth charge that transfers across a fourthsensor array at a fourth charge speed.
 27. The method of claim 25,wherein scanning the sample comprises moving the sample stage at aconstant speed.
 28. The method of claim 25, further comprising storing adata representation of said image of said sample in a computer readablememory.
 29. The method of claim 25, wherein the sample comprises anarray having a plurality of individual sites.
 30. The method of claim25, wherein said sample stage comprises a microplate array comprising aplurality of wells separated from each other by interstitial regions,wherein one or more wells includes the sample.
 31. The method of claim25, wherein the method further comprises obtaining a two-dimensional orthree-dimensional picture, image, video, or other representation of thephysical form or structure of the sample.
 32. The method of claim 30,wherein said microplate array comprises 2, 4, 6, 12, 24, 48, 96, 384 or1536 wells.